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UNIVERSITY OF GHANA
COLLEGE OF BASIC AND APPLIED SCIENCES
EXTRACTION AND CHARACTERIZATION OF CELLULOSE NANOCRYSTALS
FROM TWO LOCAL PLANT MATERIALS
BY
FAITH ZANU
(10286341)
A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN PARTIAL
FULFILLMENT OF THE AWARD OF DEGREE OF MASTER OF PHILOSOPHY IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY
JULY 2019
University of Ghana http://ugspace.ug.edu.gh
UNIVERSITY OF GHANA
COLLEGE OF BASIC AND APPLIED SCIENCES
EXTRACTION AND CHARACTERIZATION OF CELLULOSE NANOCRYSTALS
FROM TWO LOCAL PLANT MATERIALS
BY
FAITH ZANU
(10286341)
A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN PARTIAL
FULFILLMENT OF THE AWARD OF DEGREE OF MASTER OF PHILOSOPHY IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY
JULY 2019
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DECLARATION
I, FAITH ZANU, declare that I have personally, under supervision, undertaken this research
herein submitted.
Signed: Date: 25/08/2020
MPhil Candidate: Faith Zanu (10286341)
I declare that I have supervised the student in undertaking this project submitted herein and
confirm that the student has my permission for presentation and assessment.
Signed: ………………………………… Date:……25…/08…/2…02…0 ………
Principal Supervisor: Dr. Enock Dankyi
Department of Chemistry, University of Ghana
Signed: …………………………… Date: 25/08/2020
Co – Supervisor: Dr. Vitus Apalangya
Department of Food Processing Engineering, University of Ghana
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DEDICATION
This work is dedicated to God Jehovah Almighty and my dear family.
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ABSTRACT
Cellulose is a linear homopolysaccharide of repeating D-glucopyranose units which form about
15 – 20 % of the dry weight of plant biomass. It is the world’s most abundant renewable natural
polymer with unique properties such as high strength, biocompatibility, low density and
excellent mechanical properties. These excellent characteristics of the isolated cellulose
nanocrystals coupled with their wide availability enable their potential application in areas of
packaging materials to replace fossil-fuel based materials, in optical sensors, paints, and as
reinforcements in composite materials. In this work, cellulose nanocrystals were isolated from
two local plant biomass, Acasia sp. (sawie), and Palmae sp. (keteku), through a series of acid
and alkaline hydrolysis to get rid of lignin, hemicellulose and other impurities, leaving
crystalline cellulose nanocrystals. Cellulose nanocrystals were characterized by Fourier
Transform Infra-Red (FT-IR) Spectroscopy, Optical Microscopy, Scanning Electron Microscopy
(SEM), X-Ray Diffraction (XRD) and Thermogravimetric Analysis (TGA). SEM morphological
analysis showed slender nanosize particles of CNCs of approximately less than 10 µm. FTIR
analysis confirmed a removal of lignin and hemicellulose due to the disappearance of peaks at
1230 cm-1 (C – O bending) and 1765 – 1715 cm-1 (C = O stretching of aldehyde) respectively.
Peak at 1160 cm-1 showed the presence of sulphonated groups which was evidenced by the
uniform dispersibility of CNCs in solution due to the repulsive forces. The crystallinity index at
2Ꝋ (18o – 25o) was approximately -163.04 % for acacia sp. and -5460 % for palmae sp.
Thermogravimetric analysis of both acacia sp. and palmae sp. showed high thermal stability of
approximately 363.8 oC and 336.3 oC respectively. The characterized CNCs generally exhibited
outstanding properties of high crystallinity, thermal stability and tunable surfaces enabling even
dispersion in aqueous solution.
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ACKNOWLEGEMENT
My profound gratitude goes to Dr. Enock Dankyi and Dr. Vitus Apalangya for their guidance,
corrections and patience in making this work successful.
I am grateful to the lecturers, staff and colleagues of Department of Chemistry especially Mr.
Bob Essien and Mr. Samuel Owusu.
Many thanks to my colleagues, Peter Osei, Richard Owusu and Pascal Tofah for their support.
I also appreciate WACCBIP, Banga and Dr. Salifu Ali Azeko of Worcester Polytechnic Institute
for their immense help in bringing this work to a completion.
A very big thank you to the Youth Outreach family of the Global Evangelical Church, my best
friend Enoch Akoto and Mr. Jeffery Brown for their tremendous help.
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TABLE OF CONTENTS
DECLARATION………………………………………………………………………………...i
DEDICATION…………………………………………………………………………………..ii
ABSTRACT…………………………………………………………………………………….iii
ACKNOWLEDGEMENT…………………………………………………………………........iv
TABLE OF FIGURES ................................................................................................................... xi
LIST OF TABLES ........................................................................................................................ xii
CHAPTER ONE ............................................................................................................................. 1
1.0 INTRODUCTION ................................................................................................................. 1
1.1 Background ........................................................................................................................ 1
1.2 Problem Statement ............................................................................................................. 5
1.3 Justification ........................................................................................................................ 6
1.4 Aim .................................................................................................................................... 6
CHAPTER TWO ............................................................................................................................ 7
2.0 LITERATURE REVIEW ...................................................................................................... 7
2.1 Introduction ........................................................................................................................... 7
2.2 Cellulose ................................................................................................................................ 8
2.2.1 Chemistry of cellulose ........................................................................................................ 8
2.2.2 Sources of cellulose ............................................................................................................ 9
2.2.2.1 Plant/Agricultural residue source of cellulose ........................................................... 10
2.2.2.2 Bacterial source of cellulose ...................................................................................... 11
2.2.2.3 Algae source of cellulose ........................................................................................... 11
2.2.2.4 Tunicate ..................................................................................................................... 11
2.2.3 Forms of cellulose ............................................................................................................ 12
2.2.3.1 Cellulose nanofibrils .................................................................................................. 12
2.2.3.2 Nanocellulose crystals ............................................................................................... 12
2.2.4 Properties of CNCs........................................................................................................... 13
2.2.4.1 Mechanical properties ................................................................................................ 13
2.2.4.2 Physical properties ..................................................................................................... 13
2.2.4.3 Rheological properties ............................................................................................... 14
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2.2.4.4 Chemical properties ................................................................................................... 14
2.2.4.5 Liquid crystalline properties ...................................................................................... 15
2.3 Hemicellulose ...................................................................................................................... 15
2.3.1 Chemical Structure of Hemicellulose .............................................................................. 16
2.3.1.1 Xylans ........................................................................................................................ 16
2.6.1.2 Mannans..................................................................................................................... 17
2.6.1.3 Beta – glucans ............................................................................................................ 17
2.6.1.4 Xyloglucans ............................................................................................................... 17
2.4 Lignin .................................................................................................................................. 18
2.7.1 Chemical composition and structure of lignin .............................................................. 18
2.5 Inorganics ............................................................................................................................ 19
2.6 Proteins ................................................................................................................................ 19
2.7 Extractives ........................................................................................................................... 19
2.8 Nanocellulose extraction ..................................................................................................... 20
2.8.1 Mechanical treatment ................................................................................................... 20
2.8.2 Base hydrolysis ............................................................................................................. 20
2.8.3 Bleaching process ......................................................................................................... 21
2.8.4 Acid hydrolysis ............................................................................................................. 21
2.9 Characterization techniques. ............................................................................................... 23
2.9.1 FTIR.............................................................................................................................. 23
2.9.2 SEM .............................................................................................................................. 24
2.9.3 TEM .............................................................................................................................. 24
2.9.4 TGA .............................................................................................................................. 24
2.9.5 XRD .............................................................................................................................. 25
2.10 Factors affecting isolation of CNC.................................................................................... 26
2.10.1 Concentration of Acid and Base ................................................................................. 26
2.10.2 Temperature ................................................................................................................ 26
2.10.3 Time ............................................................................................................................ 27
2.10.4 Type of acid ................................................................................................................ 28
2.10.5 Type of base................................................................................................................ 28
2.11 Applications of Nanocellulose .......................................................................................... 28
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2.11.1 Wastewater treatment .................................................. Error! Bookmark not defined.
2.11.2 Biomedical application ................................................ Error! Bookmark not defined.
2.11.3 Reinforcement ............................................................. Error! Bookmark not defined.
2.12 Cellulose nanocrystal studies in Ghana .......................... Error! Bookmark not defined.
CHAPTER THREE ...................................................................................................................... 30
3.0 EXPERIMENTAL .............................................................................................................. 30
3.1 Containers and cleaning process ......................................................................................... 30
3.2 Reagents .............................................................................................................................. 30
3.2.1 Preparation of alkaloids testing and screening for alkaloids ........................................ 30
3.2.2 Preparation of 2, 4-Dinitrophenylhydrazine solution. .................................................. 31
3.2.3 Preparation of Iron (II) chloride solution ..................................................................... 31
3.2.4 Preparation of Potassium ferrocyanate solution ........................................................... 31
3.2.5 Sodium hydroxide (4 %w/w) in a 250 mL volumetric flask ........................................ 32
3.2.6 Sulfuric acid (64 %w/w) ............................................................................................... 32
3.3 Sampling of plant materials................................................................................................. 32
3.3.1 Acacia sp....................................................................................................................... 32
3.3.2 Palmae sp. ..................................................................................................................... 33
3.4 Isolation of nanocellulose crystals ...................................................................................... 34
3.4.1 Milling .......................................................................................................................... 34
3.4.2 Base hydrolysis ............................................................................................................. 34
3.4.3 Bleaching ...................................................................................................................... 34
3.4.4 Acid hydrolysis ............................................................................................................. 35
3.5.5 Dialysis ......................................................................................................................... 35
3.4.6 Sonication ..................................................................................................................... 35
3.4.7 Freeze drying ................................................................................................................ 36
3.5 Sample analyses .................................................................................................................. 36
3.5.1 Test for Alkaloids: ....................................................................................................... 36
3.5.2 Test for steroids: ........................................................................................................... 37
3.5.3 Test for Flavonoids (NaOH Test) ................................................................................. 37
3.5.4 Test for Polyphenolic compounds ................................................................................ 37
3.5.5 Test for saponins ........................................................................................................... 37
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3.5.6 Test for tannins ............................................................................................................. 38
3.5.7 Test for Terpenes .......................................................................................................... 38
3.5.8 Test for terpenoids ........................................................................................................ 38
3.6 Characterization of nanocellulose crystals .......................................................................... 38
3.6.1 Optical microscopy analysis ......................................................................................... 39
3.6.2 SEM .............................................................................................................................. 39
3.6.3 XRD .............................................................................................................................. 39
3.6.4 FT-IR ............................................................................................................................ 40
3.6.5 TGA .............................................................................................................................. 40
3.7 Quality assurance (QA) and quality control (QC) .............................................................. 41
CHAPTER FOUR ......................................................................................................................... 42
4.0 RESULTS AND DISCUSSION ......................................................................................... 42
4.1 Alkali Treatment ................................................................................................................. 42
4.2 Bleaching Treatment ........................................................................................................... 43
4.3 Acid Treatment .................................................................................................................... 44
4.4 Phytochemical screening ..................................................................................................... 45
4.5 Characterization of isolated CNCs ...................................................................................... 46
4.5.1 FTIR Analysis............................................................................................................... 46
4.5.2 SEM .............................................................................................................................. 54
4.5.2.1 Acacia sp.................................................................................................................... 55
4.5.2.2 Palmae sp. .................................................................................................................. 57
4.5.3 XRD .............................................................................................................................. 61
4.5.3.1 Untreated Acacia sp. .................................................................................................. 62
4.5.3.2 Base hydrolysis .......................................................................................................... 63
4.5.3.3 Bleaching ................................................................................................................... 63
4.5.3.4 Acid hydrolysis .......................................................................................................... 63
4.5.3.5 Cellulose nanocrystals ............................................................................................... 63
4.5.3.6 Palmae sp. .................................................................................................................. 64
4.5.3.7 Untreated ................................................................................................................... 64
4.5.3.8 Bleaching ................................................................................................................... 65
4.5.3.9 Acid hydrolysis .......................................................................................................... 65
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4.5.3.10 Cellulose nanocrystals ............................................................................................. 65
4.5.4 TGA .............................................................................................................................. 67
4.5.4.1 Acacia sp.................................................................................................................... 68
4.5.4.2 Base hydrolysis .......................................................................................................... 69
4.5.4.3 Bleaching ................................................................................................................... 69
4.5.4.4 Acid hydrolysis .......................................................................................................... 70
4.5.4.5 Cellulose nanocrystals ............................................................................................... 70
4.5.4.6 Palmae sp. .................................................................................................................. 72
4.5.4.7 Bleaching ................................................................................................................... 73
4.5.4.8 Acid hydrolysis .......................................................................................................... 73
4.5.4.9 Cellulose nanocrystals ............................................................................................... 73
CHAPTER FIVE .......................................................................................................................... 76
5.0 CONCLUSION AND RECOMMENDATION .................................................................. 76
5.1 CONCLUSION ................................................................................................................... 76
5.2 RECOMMENDATION ...................................................................................................... 77
REFERENCES ............................................................................................................................. 78
APPENDICES .............................................................................................................................. 94
APPENDIX A ........................................................................................................................... 94
FTIR spectra of acacia sp. ..................................................................................................... 94
FTIR spectra of palmae sp. .................................................................................................... 97
APPENDIX B ......................................................................................................................... 101
TGA THERMOGRAPHS ....................................................................................................... 101
TGA of Acacia sp. ............................................................................................................... 101
TGA of Palmae sp ............................................................................................................... 106
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TABLE OF FIGURES
Figure 1.1: Chemical composition of cellulose showing repeating units of glucose linked through
1,4-β-glycosidic linkage………………………………………………………………………….2
Figure 2. 1: The chemical structure of cellulose, made of cellobiose covalently linked with β (1–
4) glycosidic bond………………………………………………………………………………..9
Figure 2. 2: Mechanism of acid hydrolysis of cellulose using hydrochloric acid……………….22
Figure 2. 3: Mechanism of acid-catalyzed hydrolysis of cellulose using sulphuric acid………..22
Figure 3. 1: Photographs of (A): raw acacia sp. and (B): powdered acacia sp………………….33
Figure 3. 2: Photograhs of (A):raw palmae sp. and (B): powdered palmae sp…………………33
Figure 4. 1: Reaction mechanism of the base hydrolysis using sodium hydroxide ...................... 43
Figure 4. 2: Reaction mechanism of acid treatment using sulfuric acid ..................................... 458
Figure 4. 3: FTIR of acacia sp. from various treatment processes ............................................... 48
Figure 4. 4: FTIR of palmae sp. from various treatment processes ........................................... 496
Figure 4. 5: SEM image of the CNCs from Acacia sp. (CNC - P, scale bar = 100 µm) .............. 56
Figure 4. 6: SEM image of the cellulose nanocrystals from Acacia sp. (CNC - P, 50x, 500x,
750x, 1000x, scale bar = 10 µm) ................................................................................................ 579
Figure 4. 7: SEM image of the CNCs from palmae sp. (CNC - P, 50x, 100x, 200x, scale bar =
100 µm) ......................................................................................................................................... 59
Figure 4. 8: SEM image of the cellulose nanocrystals from Palmae sp. (CNC - P,100x, 200x,
500x, 1000x, 2000x, scale bar = 10 µm) ...................................................................................... 60
Figure 4. 9: SEM image of the cellulose nanocrystals from Palmae sp. (CNC-P, 5000x, 2000x,
200x, scale bar = 1 µm, 10 µm, 100 µm) ..................................... Error! Bookmark not defined.6
Figure 4. 10: The X-ray diffractograms of acacia sp. (a) untreated (b) bleached (c) acid
hydrolyzed and (d) CNC. ............................................................................................................ 616
Figure 4. 11: X-ray diffractograms CNC from palmae sp. (P) and acacia sp. (A). ................... 666
Figure 4. 12: Thermogram of acacia sp., alkaline treated, bleached, acid hydrolysed and CNC 722
Figure 4. 13: Thermogram of palmae sp., bleached, acid hydrolysed and CNC ........................ 755
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LIST OF TABLES
Table 2. 1: Relative amount (%) and degree of polymerization of major hemicelluloses in
different softwood and hardwood species……………………………………………………....16
Table 2. 2: Diverse acid hydrolysis for extraction of CNCs (Tang et al., 2015)………………..23
Table 3. 1: Summary of sequential extraction procedure……………………………………….36
Table 4. 1: Phytochemical screening of the raw palmae sp. and acacia sp., CNCs from palmae
sp. and acacia sp. and Standard CNC. .......................................................................................... 46
Table 4. 2: FTIR vibrational frequency and peak assignment for palmae sp. and acacia sp. ....... 51
Table 4. 3: Vibrational frequency and peak assignment of palmae sp. using Amonium persulfate
method of extraction. .................................................................................................................... 53
Table 4. 4: Crystallinity index (%) of untreated, base treated, bleached, acid hydrolyzed and
CNC of acacia sp. ......................................................................................................................... 62
Table 4. 5: Crystallinity index (%) of untreated, bleached, acid hydrolyzed and CNC from
Palmae sp. ..................................................................................................................................... 64
Table 4. 6: Amount of Weight loss (%) and Charred residue (%) for different samples of Acacia
sp. .................................................................................................................................................. 71
Table 4. 7: Amount of Weight loss (%) and Charred residue (%) for different samples of Palmae
sp ............................................................................................................................................... 7575
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LIST OF ABBREVIATIONS
CNC Cellulose Nanocrystals
CNC-A Cellulose nanocrystals from Acacia sp.
SAB Acacia sp. after base hydrolysis
SABL 1 Acacia sp. after first bleaching
SABL 2 Acacia sp. after second bleaching
SAA Acacia sp. after acid hydrolysis
CNC-P Cellulose nanocrystals from Palmae sp.
KAB Palmae sp. after base hydrolysis
KABL 1 Palmae sp. after first bleaching
KABL 2 Palmae sp. after second bleaching
KAA Palmae sp. after acid hydrolysis
CNF Cellulose Nanofibers
CNW Cellulose Nanowhiskers
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CHAPTER ONE
1.0 INTRODUCTION
1.1 Background
During the past few years, the use of renewable resources to meet the increasing human demands
of the world has received much attention. One of such environmentally friendly renewable
resources is cellulose which is considered as the most abundant polymer with a broad range of
industrial applications. It is estimated that about 7.5x1010 tons of cellulose is manufactured per
annum on a global scale (Beltramino et al., 2015).
Cellulose is a polyglucose with beta 1:4 glycosidic linkages (Figure 1.1) which make the
polymer linear and does not coil into a helical structure. It contains about 3000 monomers and
has a molecular weight of about 500 atomic mass unit (Lu & Hsieh, 2010). Individual strands of
cellulose tend to align with one another and are connected by strong hydrogen bonds which
make cellulose insoluble in water, rigid and a fibrous polymer. Cellulose materials constitute the
major component of plant fiber; 40 – 50 %. The rest is composed of hemicellulose; about 20 – 30
% and lignin; 10 – 20 % (Kargarzadeh et al., 2012; Taflick et al., 2017). Additionally, cellulose
possesses several hydroxyl groups that could be functionalized for varied products and purposes.
Cellulose may be obtained in the form of nanomaterials which are considered more suitable for
easy fabrication into other products and application.
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Figure 1.1. Chemical composition of cellulose showing repeating units of glucose linked
through 1,4-β-glycosidic linkage.
Two major cellulosic nanomaterials that can be derived from cellulose are cellulose nanofibrils
(CNFs) and CNCs (Postek et al., 1997), which are isolated from plant sources; cotton, biomass,
wood, bacteria and some sea living organisms, e.g. tunicates (Cherian et al., 2008). CNFs are
relatively longer than CNCs and are usually derived from strong mechanical fibrillation of
lignocellulosic fibers. On the other hand, CNCs are cellulose-based materials or particles that can
be isolated via acid treatment from a wide range of natural biodegradable materials (Grishkewich
et al., 2017; Postek et al., 1997).
In recent years, there has been much research on the isolation of CNCs via acid hydrolysis and
the investigation of their unique physicochemical properties and possible industrial applications
in aviation, plastic, automobiles, packaging and sensors (E. Csiszar & Nagy, 2017). The acid
hydrolysis breaks down the inter- and intra-molecular hydrogen bonds to create hydrophilic
facile surfaces which can be modified for several industrial applications. In most cases, sulfuric
acid and hydrochloric acid are employed. However, the use of hydrochloric acid produces
unstable colloidal particles, and hence its application is limited (H. Yu et al., 2013). On the other
hand, sulfuric acid used in the hydrolysis has relatively higher water consumption. Sulfuric acid
treatment is used for the degradation and displacement of the amorphous or non-crystalline
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components of cellulose through a hydrolytic breakdown. Both hydrochloric and sulfuric acid
affect the type of nanocellulose particles in structure and morphology. Different sources of CNCs
also have different dimensions of length and width sizes which eventually result in varied aspect
ratios (ratio of length to width size). The rod-like CNCs particles have an approximate width of 3
– 20 nm and length 50 – 2000 nm (Nguyen et al. 2016). For example, Cotton rod-like CNCs
have dimensions of 100 – 390 nm length and 7 – 15 nm diameter whereas flax CNC is estimated
at 100 – 500 nm length and 10 – 30 nm diameter (Postek et al., n.d.). Cellulose nanoparticles
from wood produces a lateral size of 3 – 5 nm and length 100 – 300 nm whereas CNCs from
tunicates are 15 – 30 nm lateral and 1000 – 1500 nm length (E. Csiszar & Nagy, 2017).
The CNCs, popularly referred to as nanowhiskers show some specific unique characteristics such
as high thermal steadiness up to ~300 oC, stiffness (~150 GPa), tensile stress (approximately 7.5
GPa), aspect ratio (~10 – 100), low coefficient of thermal expansion (~1ppm/K), density (~1.4
g/cm3), liquid crystalline action in suspension (Postek et al., 1997). Other chemical and physical
characteristics are large surface area (~250 m2/g), surface modification of hydroxyl group and
excellent colloidal stability (A. Csiszar et al., 2000; Tang et al., 2015). These unique properties
are characterized by employing varied instrumentations such as laser diffraction (LD), TEM, zeta
potential analyzer, UV-visible spectroscopy, SEM, XRD, tensile tester and TGA.
Due to the existence of several hydroxyl groups on the CNCs surface, many modifications are
feasible because of the high reactivity of the hydroxyl group. These modifications are done using
compounds such as silver nanoparticles, porphyrin, polymers, rosins, lectin, and functional
groups that bind with the matrixes (Hajlane et al., 2017). These unique features enable their use
in a wide variety of applications such as, wastewater treatment, biomedical, electronics, energy
and sensors. For example, recent works on the alteration of the CNC surfaces have seen their
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applications in the biomedical sector as antibacterial and anti-viral agent, biocatalyst scaffolds,
biomarkers or sensors, tissue engineering scaffolds, gene vectors and drug delivery vectors
(Barud et al., 2015). CNCs also have low or no toxicity effect when used in humans and hence
their usage in inhalers (Muthulakshmi et al., 2017).
Morphological and mechanical properties of CNCs also make them good substrates for
fabrication into matrixes to improve their mechanical properties such as high Young modulus of
78 MPa for a 5 %wt of nanocellulose fillers. In some instances, CNCs have been considered as
good replacement materials for synthetic petroleum-based materials due to the quest for bio-
based and eco-friendly nanocomposites that have arisen in the world for the past two decades.
CNCs have great potentials in their application as reinforcement agents due to their excellent
mechanical and barrier properties. Additionally, the transparent nature of CNC makes them
suitable for use in paper products and packaging material to reduce the humidity effect on paper
(F. Huang et al., 2017). Furthermore, CNCs are good potential particles to be applied as
nanocomposite fillers, reinforcements, in molecular biology and regenerative medicine (Tan et
al., 2015). Biomedically, in a study by Moreno et al. (2016), CNCs composites of protein
binding serve as good substitutes for biosensors and cell supports (Moreno et al., 2016). CNCs as
a green and cheap material are also applied in many areas including, particular enzyme
immobilization, medical materials, emulsion stabilizers, biosensors and drug delivery. In this
study, two locally grown plants, namely acacia sp. and palmae sp. were investigated as sources
of cellulose nanocrystals for potential applications in industries.
The acacia sp. is a local plant popularly grown in the Eastern region of Ghana. It is called
Akutsa in Ewe and Sapo or Sawie in Asante Twi. They are usually beaten from hard woods. The
acacia sp. has length of about 100 cm and diameter 2 cm. Its natural colour is yellow or pink and
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may have high level of lignin which impart on the colour of plant. Some decades ago, acacia sp.
was very useful when used as a sponge in bathing and washing plates. However, modernity has
made it to be of less importance in Ghana due to the introduction of synthetic sponges. Most
Ghanaians don’t patronize it anymore as sponge. Some people also use it as chewing stick but
now have less function due to the use of toothpaste and toothbrush. Currently, there has not been
any known research work on acacia sp. The acacia sp. was selected due to its physical strength,
low cost and availability. It also has the ability to be bent into different shape without breaking.
The second plant used is the palmae sp. It is popularly called Keteku in Ewe, one of the
Ghanaian south-eastern local tribes. It is also known as Afie in Asante Twi, because of its use as
a cane for punishing students. The palmae sp. is usually pinkish in colour with lots of colour
pigment called lignin. Palmae sp. varies in sizes from few millimeters in diameter and about 300
- 400 centimeters in length. It is usually grown around central part of Ghana and it’s a non-fruit
producing plant which is not grown for food. Keteku is widely used in Accra in weaving basket,
hats and mats and strong for rope. The palmae sp. was also selected for its physical strength,
bendability, low cost, and availability. Research has shown that there has not been any known
study on both the acacia sp. and palmae sp. plants in Ghana and hence the need for this studies.
1.2 Problem Statement
About 40 – 50 % of plant biomass is constituted by cellulose from which cellulose nanocrystals
are extracted. These extractions of CNCs are affected by the type of plants, environment and
various methods of isolation. As a result, the aspect ratio of CNCs varies from one geographical
location to the other. In Ghana, there are many plant and biomass materials that have these
potentials of providing a good source of cellulose for the production of CNCs through base and
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acid hydrolysis. However, little or no work has been done on the local plant biomass in Ghana as
far as the isolation of CNCs is concerned. In this study, two novel local plant materials namely
acacia sp. and palmae sp. have been selected as the major source of the extraction of CNCs in
Ghana.
1.3 Justification
Even though there exist many biomass in Ghana, not much has be done in using them as
potential starting materials for the isolation of cellulose nanocrystals. This work therefore seeks
to extract CNCs from two local plant materials, namely acacia sp. and palmae sp. that have not
been worked before. Additionally, since the environment, type of plants and mode of extraction
affect the aspect ratio and for that matter the physicochemical properties of the isolated CNCs,
this work would serve as a good ground to compare these isolated CNCs with other standards.
1.4 Aim
The main purpose of this project is to isolate cellulose nanocrystal from two local plant materials
and compare their physicochemical properties which can be used as a reinforcement material in
biodegradable films comparable to petrochemical-based packaging films.
1.4.1 Objectives
The specific objectives are:
• to extract CNCs from two local plant materials, acacia sp. and palmae sp.
• to characterize cellulose nanocrystals using SEM, XRD, FTIR and TGA
• to compare the physicochemical properties of the extracted CNCs
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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Introduction
Cellulose is one of the most important components of plant cell wall. It is a hard, fibrous and
water-insoluble polymer which plays a vital role in preserving the structure of plant cell walls (J.
George et al., 2014; J. George et al., 2011). It forms about 15 – 20% of the dry weight of plant
cell wall and is present in greater amount in secondary cell wall than primary cell wall. Cellulose
fibers in the cell wall of plants are surrounded by two main components namely lignin and
hemicellulose coupled with other materials such as ash and with an amorphous and crystalline
domain. It is a homopolysaccaharide of repeated D-glucopyranose units made up of carbon,
hydrogen, oxygen with a general formula of (C6H10O5)n (Razalli et al., 2017), where ‘n’ refers to
degree of polymerization, DP. Depending on the DP, there are three main type of cellulose,
namely alpha (α), beta (β) and gamma (γ). Alpha, beta and gamma type of cellulose have degree
of polymerization of more than 200, between 10 and 200 and below 10 respectively. Alpha
cellulose dissolves faster in about 16.5% NaOH at 20 oC followed by beta and gamma (Ritter,
1929).
The alpha cellulose is usually used to express the highest form of purity of cellulose. Different
plant species have different types and level of cellulose in their cell wall. Plant sources of
cellulose include cotton, bamboo, wood, agricultural waste, maize and biomass. Bacteria and
other sea living organisms like tunicate are also great sources of cellulose. Among all these
sources, cotton has been discovered as the purest source of cellulose. Studies have also shown
that depending on the climatic conditions and soil types for plant growth, the percentage of
cellulose may differ. Thus the cellulose content of plants in tropical areas may be different from
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those in temperate regions. Cellulosic materials are of substantial demands and importance due
to the distinctive properties of the nanocellulose crystals with their varied green and sustainable
application in industries. Because of these special characteristics, many scientists have developed
key interest in working on cellulose.
2.2 Cellulose
2.2.1 Chemistry of cellulose
The component of cellulose is found to be homopolysaccharide of anhydroglucose units joined
by glycosidic linkage bonds with repeated cellobiose as the coupling units. Figure 2.1 shows the
chemical component of cellulose made of repeated unit of cellobiose joined covalently by β-1,4
glycosidic bonds (Johnsy George, 2012; J. George et al., 2014; Johnsy George et al., 2011;
Johnsy George et al., 2012). The cellobiose units are composed of two β-D-
anhydroglucopyranose units bonded by the 1-4 glycosidic bond. This is shown in Figure 2.1.
Cellulose has three hydroxyl groups for intra and inter hydrogen bonding which gives the rigid
and tough property of cellulose in plant cell walls. This interaction of the three hydroxyl groups
of the glucose units form microfibril or elementary fibril of dimensions, 3- 4nm in diameter and
1-2 um in length. Microfibrils of diameter 10 -15 nm entwine into a network to form fibril and
this makes them stronger than steel of same length. (Du et al., 2017; Y. Wang et al., 2006). The
microfibrils combine to form macrofibrils of diameter 15 to 16 nm and length, few micrometers
which are later packed into cellulose fibres of diameter 20 to 50 um and length 1 to 4 mm
(Lavoine & Bergström, 2017). Each microfibril of cellulose contains nanocrystalline rod-like
fragments which is referred to us cellulose nanocrystals or cellulose nanowhiskers which have
regular arrangement of anhydrous glucose. This regular arrangement known as micelle also
makes cellulose crystalline, rough and rigid. (Du et al., 2017) The crystalline part of cellulose is
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a major part of cellulose made up of regular arrangement of beta-anhydroglucose units joined
together by 1-4 glycosidic linkage. This regular arrangement makes cellulose exhibit crystalline
nature with unique physical properties.
The amorphous part of cellulose is the non-crystalline part of cellulose with irregular structured
arrangement of anhydrous glucose via glycosidic bonds. This irregular arrangement enables them
to be removed by acid hydrolysis (Cherian et al., 2011; Cherian et al., 2010).
Figure 2. 2 The chemical structure of cellulose, made of cellobiose covalently linked with β
(1–4) glycosidic bonds.
2.2.2 Sources of cellulose
The main source of cellulose is plants. Others include algae, bacteria, and some sea living
organism known as tunicate. Many cellulosic materials have been used to produce nanocellulose
crystal over the past few years. The source of cellulose is one of the vital factors that affect the
shape and structure of nanocellulose crystals. Each cellulosic material produces its own unique
form of nanocellulose crystals in length, diameter, and morpholology. Different materials have
different level of cellulose which eventually affect the yield of CNC produced. Example of
materials used are cotton (E. Csiszar & Nagy, 2017; de Morais Teixeira et al., 2010; Yang et al.,
2018), pineapple (Cherian et al., 2011; R. B. Santos et al., 2013; R. M. d. Santos et al., 2013),
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rice straw (Ahmadi et al., 2015; Lu & Hsieh, 2010, 2012), corn (S. Huang et al., 2017; Yu et al.,
2014), sugarcane bagasse (de Oliveira et al., 2016), coconut husk (M. F. Rosa et al., 2010) soy
(Neto et al., 2016; Neto et al., 2013), potato (D. Chen et al., 2012), garlic (Kallel et al., 2016)
and tomato (Jiang & Hsieh, 2015). The following are some of the major sources of cellulose.
2.2.2.1 Plant/Agricultural residue source of cellulose
Plant sources of cellulose are cheap and readily available. Different parts of plants including
stem, leaf and fruit serve as the major source of cellulose. Some of the plants include jute, ramie,
sisal, flax, hemp, cotton and wood (E. Csiszar & Nagy, 2017; de Morais Teixeira et al., 2010;
Yang et al., 2018). Wood is a major source of cellulose, (B. Li et al., 2015; Moriana et al., 2016;
M. F. Rosa et al., 2010) but Cotton contains the highest form of pure cellulose, 95 – 97% with a
diameter fibril of 500 nm and longitudinal length of few microns with a high crystallinity. (E.
Csiszar & Nagy, 2017; de Morais Teixeira et al., 2010; Morais et al., 2013). This is ascribed to
the low noncellulosic parts of cotton cellulose when contrasted to wood. Studies have shown that
most agricultural biomass such as wheat, sawdust, oil palm, corn straw, rice straw, sugarcane
bagasse, sugar beet, jute, pineapple, bamboo, mengkuang leaves and cotton stables produce an
appreciable level of cellulose (Cherian et al., 2011; Khoshkava & Kamal, 2014; Z. Li et al.,
2016). However, wood is a fetching source of cellulose because of its relative abundance on
earth. Plant materials are usually strong, stiff, tough and low in density with high proportion of
cellulose.
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2.2.2.2 Bacterial source of cellulose
Bacteria cellulose is made by microbial actions of certain bacteria species. Some examples of
these species include Komagataeibacter xylinus and K. xylinus during vinegar fermentation.(J.
George et al., 2014; J. George et al., 2011; Johnsy George et al., 2012). The most common
bacteria species used is the Gluconacetobacter xylinus. These bacteria species produce thick gel
usually with high volume of water, 97 – 99 mL in a well culturing environment. Bacteria
cellulose are also considered as one of the purest form of alpha cellulose devoid of hemicellulose
and lignin. (Chi & Catchmark, 2017; Mohammadkazemi et al., 2015; Nogi & Yano, 2008;
Shankar et al., 2018). The degree of polymerization is relatively high in bacterial cellulose,
between 2000 and 6000. Even though different sources of cellulose produce different
characteristic nanocellulose crystals, research shows that both bacterial and wood sources have
similar properties. However, these pure form may cause contamination in the alimentary carnal
of humans. (M. George et al., 2017)
2.2.2.3 Algae source of cellulose
The cell wall of many algae is composed of highly crystalline cellulose. Red, green and yellow
algae are the main types of algae that produce cellulose in moderately high quantity. Of these
three types, green algae are considered to be the most favorable (Mihranyan, 2011).
2.2.2.4 Tunicate
The cellulose is found in their thick skeletal mantle which covers the epidermis of tunicate.(J.
George et al., 2014). It is produced by enzyme action in the membrane of the epidermis and may
vary from one species to another. The cellulose produced in the outer skin of tunicates are called
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tunic. These tunics are used to form the pure form of cellulose referred to us tunicin. Different
species of tunicate have been used to produce CNC. These include sea squirts Ascidiacea,
Halocynthia roretzi, Halocynthia papillosa. and Metandroxarpa (Cherian et al., 2010)
2.2.3 Forms of cellulose
Cellulose occurs in varying forms. Below are the forms of cellulose.
2.2.3.1 Cellulose nanofibrils
Generally cellulose nanofibrils, CNFs have dimensions of 3 to 50 nm in diameter and few
micrometers in length (Lavoine & Bergström, 2017) which is usually produced via
caboxymethylation, carboxylation, quaternation, pre-enzyme treatment or pre mechanical
treatment. The cellulose nanofibrils contain both crystalline and amorphous parts which are
further broken down into nanocellulose crystals via acid hydrolysis.
2.2.3.2 Nanocellulose crystals
Nanocellulose is a nanomaterial with distinct chemical and physical properties of low density,
renewability, biocompatibility, tunable surface chemistry of the hydroxyl groups and high
strength. In 1950 Ranby and Ribi produced the first colloidal sulphonated CNC using sulfuric
acid hydrolysis from wood and cotton (R. M. d. Santos et al., 2013). Researchers have used
several acids such as hydrochloric, phosphoric, hydrochromic and phosphotungstic acid to
extract CNCs (Tang et al., 2015).
Nanocellulose may exist as aerogel which are mesoporous material with approximate porosity of
90% (2 to 50 nm pore size). This flexible and strong aerogels can be used in sound absorption
and insulation due to their low thermal conductivity and dielectric property. In addition the ultra-
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low density, tunable surface chemistry and unique mechanical characteristics of CNC make them
favorable in constructing biomedical scaffolds, insulators, and devices for storage. (Lavoine &
Bergström, 2017).
2.2.4 Properties of CNCs
2.2.4.1 Mechanical properties
CNCs are unique in mechanical properties. They are very stiff, strong and elastic. This is as a
result of the regular arrangement of the crystalline glucose domain of cellulose. Presence of both
crystalline and amorphous domain of cellulose, make nanocrystals have different modulus. The
theoretical estimated axial modulus of CNC is approximately 50 – 170 GPa, giving a value close
to Kevlar (60 – 125 GPa) which is tougher than steel (200 – 220 GPa) (Kaushik, 2016).
The theoretical predictions of the tensile strength are approximately 0.3 – 22 GPa. High tensile
strength of nanocellulose emanate from the increased chain structure of crystalline cellulose
domain, large number of inter and intra hydrogen bonding. In addition, there is high density of
covalent bonds in CNCs which make them exhibit high tensile strength.
2.2.4.2 Physical properties
The 4-anhydro-D-glucopyranose in glucose possesses a chair conformation sequentially and is
able to rotate through 180o. The three OHs on each constituent can form high hydrogen bonds to
form slender fibre structures and semicrystalline packing which account for the high
cohesiveness of cellulose. Each nanocrystalline particle is an elongated needle-like or rod-like
structure of diameter, 5 – 30 nm and length, 100 – 500 nm. CNCs are very stiff and strong with
young modulus of approximately 139.5 + 3.5 (Wu et al., 2010).
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2.2.4.3 Rheological properties
The deformation and flow of particles is yet another unique property of CNCs. This is influenced
by the liquid crystalline nature of CNCs. Other factors influencing such properties are the spatial
ordering and gelation of the particles. Dilute suspension of CNCs also show shear thinning at
low concentration (Bercea & Navard, 2000). However, CNCs at high concentration deviate from
the original shear property. And this is due to the rod-like structure of CNCs which make them
orient at critical shear rate that destroys the chirality of the crystals. The aspect ratio also affects
the rheology of the crystals. At higher aspect ratio, the particles in CNCs tend to have high
relaxation time which favors the alignment after shear (Majoinen et al., 2012). Different acid
exhibit varying shear thinning. CNCs produced via sulfuric acid tend to have low shear thinning
where as those of hydrochloric acid have very high shear thinning. This is because, the CNC
produced via hydrochloric acid do not have chiral centers (Hasani et al., 2008).
2.2.4.4 Chemical properties
CNCs have many hydroxyl groups which enable them to be subjected to several surface
modifications with different chemical substances. The ability to undergo surface modification or
functionalization makes them have positive or negative charges for further applications. Some of
the surface functionalization includes esterification, etherification, amidation, oxidation,
carbamation, polymer grafting, silylation and nucleophilic substitution. Sulfation and
phosphorylation are the basic esterification processes which lead to formation of chiral centers
for further application. The surface modification of cellulose also enhances the production of
nanocomposites for applications in the industries such as plastics, water, biomedical engineering.
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2.2.4.5 Liquid crystalline properties
Under suitable conditions, CNCs exhibit liquid crystallinity due to the regular ordered
arrangement of the particles called a nematic phase. The rod-like structure of the CNCs particles
forms chiral centers when dispersed in water to exhibit crystallinity. Their liquid crystallinity
emanates from their hardness, aspect ratio and the capacity to orient in solution. The CNCs have
cholesteric phase or chiral nematic phase due to helical twist structure like a screw. The liquid
crystallinity property is affected by shape, electrolyte, size, charge and external stimuli factors.
Due to the liquid crystallinity property, CNCs exhibit unique bi-refrigerant property which
produces interesting optical phenomena.
2.3 Hemicellulose
Hemicellulose contributes about 20 - 30% of dry weight of soft and hardwood. It is the alkali
soluble component of plant cell wall after the removal of pectic substances. Like cellulose,
hemicellulose has degree of polymerization in the range of 50 – 300 and heteropolysacharides
constituents with their monomeric components as anhydrohexoses, anhydropentoses and
anhydrouronic acids (Table 2.1). Hemicellulose is vital in plant cell wall in crosslinking with
cellulose fibrils and lignin matrix to increase the mechanical strength of cell wall. Hemicellulose,
like lignin has low decomposition, which is made of acetyl and uronic ester groups. In the
determination of the presence of hemicellulose in a plant material using the FTIR, an eminent
peak at 1200 to 1300 cm-1 is characteristic of C-O bending vibration in the uronic ester group.
(Deepa et al., 2011; Maiti et al., 2013).
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Table 2. 3: Relative amount (%) and degree of polymerization of major hemicelluloses in
different softwood and hardwood species
Material Hemicellulose content Degree of polymerization
Softwood
Loblolly pine 15.3 ……
Black Spruce 17.4 ……
Galactogucomannan ~20 40-100
Gluconoxylan 5-10 50-185
Hardwood
Birch 33 ……
Gluconoxylan 15-30 ~200
Gluconomannan 2-5 ~70
2.3.1 Chemical Structure of Hemicellulose
Structurally, there are four main types of hemicellulose, namely; xyloglycans (xylans),
mannoglycans (mannans), β-glucans, and xyloglucans.
2.3.1.1 Xylans
Xylans, constitute the main component of hemicellulose of about 20 - 15 % in softwood (juniper,
pine, redwood and yew), 10 - 35% in hardwood (mahogany, oak, walnut and balsa) and 35 - 40%
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of the total biomass residue of some annual plants. Xylan, also called xyloglycans has two forms,
homoxylans and heteroxylans. The homo unbranched xylan comprises of β (1-4)-D-
xylopyranose joined together by (1-3, 1-4) or (1-3) glycosidic linkages which are commonly
found in seaweeds. Heteroxylans, have more complex substituted structures made of
glucuronoxylans and arabinoxylans.
2.6.1.2 Mannans
Mannans refer to the linear polymer polysaccharides of sugar mannose monomers. It is usually
found in plant yeast. Galactoglucomannan is the main hemicellulose in soft wood of about 20%.
2.6.1.3 Beta – glucans
Beta-glucans are sugars linked by β-glycosidic bonds which are usually found in cell walls of
bacteria, yeast, fungi, lichens and algae. Glycosidic linkage in beta glucan exist between carbon
1 and 3 or 1 and 6. Research shows that beta-glucans have some health benefits in treating
diabetes, HIV/AIDs, cancer, high cholesterol level, cold, skin diseases and hepatitis.
2.6.1.4 Xyloglucans
Xyloglucans are the most inexhaustible hemicellulose in the cell wall of vascular plants.
Xyloglucan often binds cellulose fibrils together and has a β1→4-linked glucose, most of which
are replaced with 1-6 linked xylose side chains. Xylose remnants are usually capped with a
galactose remnants, sometimes followed by a fructose residue. Below is the structure unit of
xyloglucans.
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2.4 Lignin
In 1813, the Swiss botanist A. P. de Candolle defined lignin as a fibrous, insipid material which
does not dissolve in water and alcohol but dissolves in weak base and can be precipitated by an
acid (de Candolle, 1813). Lignin is a complex polymer of cross-linked phenols which plays a
very important role in plants particularly, wood and bark for their rigidity because it does not rot
easily. It constituent about 20 - 35% (J. Li et al., 2011) of dry wood, 14 - 25% herbaceous plants
(Y. Chen et al., 2009) and about 30% (Boerjan et al., 2003) of non-fossil organic carbon.
Physically, lignin is very hard, compact and strong. Lignin has phenylpropanoid as repeating unit
held together by ether bond or carbon-carbon bond which enhance the hardness of plant cell
wall, as well as the mechanical support of plants via high link network (Song et al., 2013). The
phenolic compounds in lignin is the colour pigment which gives colour to each cellulosic
material. Thus an indication for the absolute elimination of lignin is the perfect change of color
in the plant materials.
2.7.1 Chemical composition and structure of lignin
Lignin is a heterogenous polymer of guaiacyl propane (G), syringyl propane (S) and
hydroxylphenyl propane (H) units linked by ether bond, carbon-carbon bond and β-O-4 ether
bond. It is a crosslink of the three main lignols which are consolidated into lignin in the structure
of phenylpropanoids. Depending on the type of plants lignin varies in the combination of the
various constituents. In the dicotyledonous angiosperm plant, G and S are present. Whereas in
monocotyledonous lignin, all the three constituent are present in approximately equal proportion.
The degree of polymerization, DP of lignin is approximately 4000 and has molecular unit above
10000 u. They are usually of aromatic polymers which are relatively hydrophobic. Lignin is
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highly cross-linked and also amorphous like hemicellulose. The ester linkage between the
hydroxyl group of lignin and carboxyl of uronic acid of hemicellulose are highly affected by
alkali treatment. Thus lignin is partly removed by base hydrolysis but more effectively removed
via bleaching using hydrogen peroxide, and sodium chlorite. Lignin however does not dissolve
in acid and therefore usually hinder acid hydrolysis. In order to facilitate the acid treatment,
delignification via bleaching is paramount for the extraction of pure nanocellulose cyrstals.
2.5 Inorganics
Another component of plant materials is inorganics which basically refers to the ash content. The
ash content represents the mineral salts and other inorganic matter like silica. Their percentage in
plant is minute but may interfere with chemical processes. Usually they are burnt and removed at
relatively high temperatures (Han & Rowell, 1997).
2.6 Proteins
Proteins form approximately 1% of the chemical component of plant materials. They are large
polymers of amino acids, enzymes, toxins and occur in three main classes namely, glycine-rich
protein, proline-rich protein and hydroproline protein. The proteins are usually connected to the
lignin in plant materials that makes them to be removed during the bleaching process.
2.7 Extractives
Extractives are made up of various monomers, dimers and polymers of waxes, resin acid,
steroids, terpenes, phenols, fatty alcohols, fatty acid and fats. They are easily removed by one of
the many extraction processes and hence the name extractives. These also refer to other non-
cellulosic components such as pectin, ash, suberin, cutin, wax and fatty substance which reduce
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water losses from cell wall. Pectin substances are easily broken by enzymes and acids to be
washed away. The extractives are also usually hydrophobic and plasticized. (Song et al., 2013).
2.8 Nanocellulose extraction
The preparation of cellulose whiskers or CNCs involves two main stages, namely, base and acid
treatments. The main aim of these treatments is to extricate lignin, hemicellulose, ashes and other
impurities. The following are the stages of extraction of nanocellulose crystals.
2.8.1 Mechanical treatment
Plant materials usually come in big and large surface areas. Many plant substances are chopped
into smaller sizes and milled into powder. Simple blender is used for soft wood plant materials.
However, in milling hardwoods, robust milling machines are used. The powdered samples are
sieved to obtain uniform surface area. This enhances the subsequent chemical treatments.
2.8.2 Base hydrolysis
The first hydrolysis process in the extraction of cellulose nanocrystrals is the alkaline hydrolysis
which requires the addition of bases such as sodium hydroxide or potassium hydroxide. This
process removes the irregular amorphous hemicellulose domain of the fiber. Usually, dilute base
of 4 or 5%wt of NaOH or KOH is enough to break into the hemicellulose structure. The alkaline
hydrolysis dissolves the hemicellulose which is washed away to a pH of 7 using a centrifuge.
Often, some extractives like terpens, resin, fats, fatty acid, waxes and toxins are removed with
the hemicellulose during the alkali treatment. (Ng et al., 2015)
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2.8.3 Bleaching process
The bleaching treatment of the cellulosic material is usually done after the base hydrolysis. The
aim of this process is to isolate the lignin component of the fiber. The lignin component which is
the color pigment of the fiber is bleached using hydrogen peroxide, glacial acetic acid and
sodium chlorite. The hydroxyl radical or chlorine radical is able to break into the aromatic
phenolic rings of the lignin and washed away.
Bleaching reaction scheme;
H2O2 H
+ + HO - 2
HO -2 OH
- + [O]
[O] + I IO
Where ‘I’ refers to the lignin materials and some impurities associated with cellulose (Ng et al.,
2015).
2.8.4 Acid hydrolysis
Acid treatment is the last stage of the hydrolysis process in the isolation of nanocellulose
crystals. Surface sulfate ester groups are often produced when sulfuric acid is used (Beltramino
et al., 2015; Du et al., 2017; J. George et al., 2011; Q. Q. Wang et al., 2012; Y. Wang et al.,
2006; H.-Y. Yu et al., 2013). Other acids commonly used are hydrochloric acid (H.-Y. Yu et al.,
2013; H. Yu et al., 2013) (Figure 2.3), oxalic acid (Deepa et al., 2011), bromic acid, formic acid
and phosphoric acid (Tang et al., 2015) (Table 2.2).
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Sulfuric acid is usually preferred to other acids because of the stability of the surface charged
nanocellulose crystals which lead to a colloidal suspension of cellulose fibers as a result of the
electrostatic negative charges from the O-SO3H (Figure 2.3). Other acids like HCl are unable to
produce surface charges during the hydrolysis process (Figure 2.2).
Figure 2. 2: Mechanism of acid hydrolysis of cellulose using hydrochloric acid
Figure 2. 3: Mechanism of acid-catalyzed hydrolysis of cellulose using sulphuric acid
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Table 2. 4: Diverse acid hydrolysis for extraction of CNCs (Tang et al., 2015)
Sources Chemicals Conditions
Old corrugated container Phosphoric acid 25oC, 6 h
Cotton Sulfuric acid 60oC, 2 h
Kenaf fiber Sulfuric acid 80oC, 4 h
Corncob Sulfuric acid 45oC, 45 min
Rice husk Sulfuric acid 50oC, 40 min
Coconut husk fiber Nitric acid 70oC, 1 h
H. Sabdariffa fiber Oxalic acid 20oC, 3 h
2.9 Characterization techniques.
In order to confirm the extracted nanocellulose crystals for further application, the following
characterization techniques are employed.
2.9.1 FTIR
The FTIR of a sample is one of the main parameters in characterizing cellulose nanocrystals. In
this characterization technique, functional groups of the isolated compound are used to determine
the intrinsic physicochemical properties of the nanocellulose crystals. For example a peak at
3400 cm-1 corresponds to a hydroxyl group on the cellulose. A close examination of FTIR
spectrum is able to determine when all hemicellulose and lignin have been removed due to the
disappearance of some important peaks from the original. In a research by Hanieh Kargarzadeh
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et al. (2012) where a study on the effects of treatment conditions on the shape, crystallinity and
thermal stability of CNCs extracted from Kenaf fibers were conducted, the FTIR spectra of the
raw fiber, after base and bleaching, confirm a complete removal of the hemicellulose
(Kargarzadeh et al., 2012).
2.9.2 SEM
Morphology, topography, composition and crystallographic information of CNC are major
characteristics that distinguish it from other materials. The rod-like (may be curled) or needle-
like shape of CNC is determined by the scanning electron microscopy, SEM. (Morais et al.,
2013). The SEM is employed because of the nano-size of the cellulose materials. It is also
employed due to its high magnification, high resolution, 3-dimensional image and larger depth of
analysis (Sheltami et al., 2012).
2.9.3 TEM
TEM is of higher resolution than SEM and gives the geometric dimensions of diameter and
length of the isolated crystals in nano-size. The TEM also help calculate the aspect ratio
(length/diameter) (Chi & Catchmark, 2017; Deepa et al., 2011; Du et al., 2017; Morais et al.,
2013; Moriana et al., 2016; Tummala et al., 2017) which determines the suitability of the
crystals as reinforcement (W. Chen et al., 2011).
2.9.4 TGA
Internal stability due to high resistivity to heat is one of the unique inherent physical
characteristics of CNC. CNCs are very stable with a high crystal structure which prevents them
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from getting decomposed easily. This intrinsic characteristic of CNCs makes it suitable for the
production of plastics for packaging. The CNC basically decompose at high temperatures of
approximately 300 oC (Deepa et al., 2011; Fortunati et al., 2014; Jung et al., 2017; Maiti et al.,
2013; Y. Wang et al., 2006)
2.9.5 XRD
X-ray diffraction is used to determine the crystallinity of cellulose by calculating the crystallinity
index, Ix of the sample. Basically, the crystallinity of cellulose increases through the extraction
stages from base hydrolysis, bleaching to acid hydrolysis because of the removal of the irregular
non-crystalline components of cellulose. X-ray diffraction is therefore able to identify the
crystalline material and the non-crystalline part. It is also used to determine the crystal structure
and the d-spacing of the constituent particles. In this process a cathode ray is generated by
heating the filament at a particular voltage to release electrons. These electrons are converted
into a monochromatic light intensity and irradiated against the samples. The monochromatic
light is therefore diffracted at a specific unique angle since each particle interacts with the light
differently. The crystallinity index is recorded and plotted against the diffraction angle. The
diffraction angular range 2Ɵ is in the range of 5 – 40o and a step time of 2.0 s
CrI = ( I200 – Iam) / I200
CrI is the crystallinity index
I200 is the maximum intensity at the 200 lattice plane
Iam is the amorphous intensity at approximately 2Ɵ = 18
o
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2.10 Factors affecting isolation of CNC
2.10.1 Concentration of Acid and Base
The concentration of the base and acid affects the CNC produced in morphology, structure, and
geometry. At high concentration, hydrolysis is very effective in breaking the glycosidic bonds of
the cellobiose to produce high crystalline nanocellulose (Ahmadi et al., 2015; Beltramino et al.,
2015; Bondeson et al., 2006). However, high concentration of the acid, over a long period of
treatment may burn the cellulose samples due to the excessive heat produced by the acid
(Bondeson et al., 2006). At low concentration, the hydrolysis may not be effective due to the low
level of protons produced to penetrate the amorphous region of cellulose. Different
concentrations produce varied diameter and length of CNC with different aspect ratio. For
example, concentrations of H2SO4 from 64, 63 and 59 %w/w produce CNCs with different
dimensions in diameter and length (Kargarzadeh et al., 2012).
2.10.2 Temperature
Basically, hemicellulose and lignin which are the main impurities associated with cellulose tend
to dissolve at high temperature of 70 to 80oC. (Eronen et al., 2011; Yoshida et al., 2008). In a
study by Yang et al. (2018), it was observed that hemicellulose, lignin and cellulose degrade at
varying temperatures due to structural changes. In effect, individual components can be isolated
depending on the temperature of the process. The base hydrolysis temperature for the removal of
hemicellulose is higher than the acid hydrolysis. Acid hydrolysis may occur at room temperature
and increase to about 70 oC depending on the concentration (Beck-Candanedo et al., 2005). At
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high concentration of acid, temperature will have to be reduced due to the excessive heat
produced via the preparation of acid solution (Kargarzadeh et al., 2012).
2.10.3 Time
Isolation of nanocellulose crystals is time dependent in the base and acid hydrolysis as well as
the one-step-ammonium persulphate (APS) process. The minimum time of base hydrolysis is 2
hours (Bondeson et al., 2006). The time of the hydrolysis depends on the concentration of the
base used. High concentration of sodium hydroxide, about 4 M requires 2 hours whereas low
concentration of about 2 M requires longer time, about six to eight hours (Cherian et al., 2010).
During the acid treatment of cellulose to remove the irregular amorphous region, the duration of
the process also influences the diameter, length and structure of CNCs. Long time (about 5
hours) of hydrolysis reduces the length of rod-like CNCs as evidenced during the extraction of
CNC from cotton (Dong et al., 2016), wood (Beck-Candanedo et al., 2005), pea hull fibers (Y.
Chen et al., 2009), microcrystalline cellulose (Bondeson et al., 2006) and coconut husk fibers
(M. Rosa et al., 2010). In this research work it was observed that the at high concentration of
acid, approximately 64 %w/w, less time of about 30 mins is required whereas low concentration
of about 2 %w/w may require a whole day. The one step Ammonium persulfate, APS process
requires about 24 hours when concentration is relatively low. However, at very high
concentrations, about 6hours is required. Comparatively, the APS method saves time as base and
acid hydrolysis process are time consuming.
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2.10.4 Type of acid
Different acids produce varying forms of nanocellulose crystals. Some of the acids that have
been used include, sulfuric acid (Beltramino et al., 2015; Du et al., 2017; Q. Q. Wang et al.,
2012; Y. Wang et al., 2006), formic acid (B. Li et al., 2015), hydrochloric acid , phosphoric acid
(Tang et al., 2015) and bromic acid. Among these acid types, sulfuric acid and phosphoric acid
have produced more stable cellulose nanocrystals due to their ester formation at the surface of
cellulose. Their nematic structure enhances their stability. In addition, the sulfate ions and
phosphate ions generate a high electrostatic force of repulsion that enhances the uniform
dispersibility of nanocellulose in solution (Beck-Candanedo et al., 2005; de Souza Lima &
Borsali, 2004). Those produced via hydrochloric acid tend to lack chiral nematic centers which
do not enhance dispersion.
2.10.5 Type of base
Sodium hydroxide has been the most widely used base in the alkaline hydrolysis process for the
synthesis of CNC due to its ready availability, cheap cost and easy to handle. Other bases that
have been used over the past decade include potassium hydroxide. The concentration of base
ranges from 2 M to 4 M which determine the length of the base hydrolysis process. Lower
concentration of base requires longer time for the removal of hemicellulose whereas high
concentration takes few hours for effective removal of hemicellulose.
2.11 Applications of Nanocellulose
CNCs have great advantage in their application because of their high mechanical strength,
surface area, availability, sustainability, renewability, biodegradability, and specific aspect ratio.
The hydroxyl groups are modified by the process such as esterification and acetylation to
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produce surface carboxylic acid, amine, protein and thiol functional group for several industrial
applications such as waste water treatment, biomedical and reinforcements in packaging
materials
2.12 Cellulose nanocrystal studies in Ghana
Studies on the extraction of CNCs from Ghanaian local plants have been very minimal. Very few
studies have been carried out on the potential application of cellulose nanocrystals in the
packaging industries, water treatment and biomedical engineering (Suopajärvi et al., 2013).
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CHAPTER THREE
3.0 EXPERIMENTAL
This chapter focuses on the detailed procedures in isolating nanocellulose crystals from two local
Ghanaian plants and their characterization.
3.1 Containers and cleaning process
All glassware and Eppendorf tubes used in the isolation of nanocellulose crystals were cleaned in
a detergent for 6 h, washed and rinsed with deionized water two times. They were later immersed
in 50 % HNO3 bath at 90
oC for a period of 24 h, rinsed several times and dried overnight.
3.2 Reagents
All chemicals used were of analytical reagent grade. Sulfuric acid, H2SO4 (98 %) was purchased
from Kosdaq Company, 30 % w/w Hydrogen peroxide, H2O2 from Qualikems Laboratory,
sodium hydroxide, NaOH pellet and sodium chorite, NaClO were obtained from the Department
of Chemistry.
Phytochemical reagents used for phytochemical tests were prepared according to the following
procedures to carry out the presence of chemical compounds such as saponins, flavonoids,
alkaloids and polyphenols which naturally occur in plant extracts.
3.2.1 Preparation of alkaloids testing and screening for alkaloids
a) Wagner’s reagent: about 1 g of iodine and 2.0 g of potassium iodide were dissolved in
water in a volumetric flask and the solution made up to 100 mL with water. The reagent
was dark-brown in colour.
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b) Dragendorff’s reagent: 4.0 g of hydrated bismuth was dissolved in 20 mL concentrated
nitric acid and the resulting solution added to a 13.5 g of potassium iodide in 25 mL of
water. Black precipitate formed was filtered off and the filtrate made up to 50 mL with
water. Reagent was orange in colour.
c) Meyer’s reagent: 1.4 g of mercury iodide in 60 mL of water was added to a 10 mL
solution of 5.0 g potassium iodide in water and made up to 100 mL with deionized water.
Reagent was pale yellow in colour.
3.2.2 Preparation of 2, 4-Dinitrophenylhydrazine solution.
3.0 g of 2, 4 dinitrophenylhydrazine was placed in a beaker and 20 mL distilled water and 70 mL
of 95 % ethanol added. Mixture was stirred, placed in ice bath and 15 mL of concentrated
sulphuric acid was added. A clear orange solution was obtained.
3.2.3 Preparation of Iron (II) chloride solution
100 mL of iron (II) chloride solution was prepared by mixing 10 mL aqueous iron (II) chloride
and 90 mL of distilled water. A clear pink solution was obtained.
3.2.4 Preparation of Potassium ferrocyanate solution
0.2 g of solid potassium ferrocyanate was dissolved in 10 mL of distilled water to obtain a clear
solution.
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3.2.5 Sodium hydroxide (4 %w/w) in a 250 mL volumetric flask
10.0 g of sodium hydroxide pellets were weighed and dissolved in a beaker of water. The
resulting solution was transferred into a 250 mL volumetric flask and topped up to the mark.
3.2.6 Sulfuric acid (64 %w/w)
64 mL of 98 % w/v H2SO4 analytical reagent grade was measured and added to a beaker of 36
mL deionized water. The solution was prepared over an icebath due to the excessive heat
produced during the preparation of the highly concentrated solution (Filson & Dawson-Andoh,
2009).
3.3 Sampling of plant materials.
3.3.1 Acacia sp.
Samples used for this research work were purchased from the Madina market in Accra and
milled at a commercial mill at Koforidua. Figure 3.1 shows the nature of the raw and milled
acacia sp.
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Figure 3. 3: Photographs of (A): raw acacia sp. and (B): powdered acacia sp.
3.3.2 Palmae sp.
Samples were purchased from the Madina market in Accra, Ghana and milled at Koforidua using
the commercial mill. Below is a picture of the raw and milled form of palmae sp.; Figure 3.2.
Figure 3.2: Photograhs of (A):raw palmae sp. and (B): powdered palmae sp.
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3.4 Isolation of nanocellulose crystals
Milling, base treatment, bleaching and acid treatment were carried out to extract CNCs from both
acacia sp. and palmae sp.
3.4.1 Milling
Samples were cut into small lengths of about 5 cm and width 2 cm with a pair of secateurs and
milled into powder with a commercial milling machine.
3.4.2 Base hydrolysis
150 mL of 4 % w/w of an already prepared sodium hydroxide, NaOH, was used to treat 5.0 g of
raw palmae sp. to remove hemicellulose. The solution was heated over a water bath at a
temperature of 70 to 80 oC for 3 h. The process was repeated for acacia sp. Pinkish colour of the
palmae sp. turned into a deep dark-brown solution whereas the yellowish color of the acacia sp.
turned into a brown solution. Solution was left to cool and washed with distilled water using a
centrifuge at 3500 rcf to a pH of 6 - 7. During washing, the colors faded gradually to light brown.
3.4.3 Bleaching
Repeated bleaching of base hydrolyzed samples was done using 30 % w/w H2O2 and NaClO.
150 mL of H2O2 was used for the first treatment for 6 h under constant stirring at 70 rpm (Table
3.1). The color of the acacia sp. changed to a pale yellow and centrifuged to decant the
supernatant. 150 mL of the NaClO was used for the second bleaching for 24 h to a white color.
Solution was centrifuged and supernatant decanted leaving the samples for acid hydrolysis. The
same steps were repeated for the palmae sp. Here the color change was from light brown to
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yellow, pale yellow and white. It was observed that the palmae sp. took more time of about 48 h
to a white coloration.
3.4.4 Acid hydrolysis
The highly concentrated sulfuric acid of 64 % w/w prepared was used to treat the bleached
samples at a temperature of 45 oC for 30 min (Table 3.1). This was done over an ice bath due to
the excessive heat produced by the acid.
3.5.5 Dialysis
In the extraction of cellulose nanocrystals, dialysis plays a vital role. In this process, residual
hydrogen ions and other excess ions are removed to present a pure CNCs. This was done for 7
days to ensure a complete removal of ions. A liter of bucket full of deionized water was used
each day. During this process, the dispersion is put in a dialysis bag and later placed in the
bucket full of water until a constant pH is obtained.
3.4.6 Sonication
Sonication is another vital treatment in the extraction of cellulose nanocrystals. In this process,
suspended sulfonated CNCs particles were forced into cloudy colloidal solution by a sonicator.
The sonicator was set at thirty second pulse and amplitude at 25 m for 10 min.
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3.4.7 Freeze drying
Freeze drying of sonicated samples were done at a very high pressure and very low freezing
temperature for three days. Samples however were first frozen and drying was done at high
pressure to suck water through sublimation. The resultant products were crystalline.
Table 3. 2: Summary of sequential extraction procedure
Steps Reagents Extraction conditions Name of Process
1 Sodium hydroxide (4% w/w) 70 oC to 80 oC for 3 h Base hydrolysis
2 Hydrogen peroxide (30 % w/w) Room temperature Bleaching
and Sodium chlorite
3 Sulfuric acid (64 %w/w) 45 oC for 30 min Acid hydrolysis
3.5 Sample analyses
3.5.1 Test for Alkaloids:
About 4 mL of the extracted cellulose nanocrystals colloidal solution was treated with 20 mL of
2.0 M HCl solution and the mixture warmed, filtered and divided into three test tubes. To each
tube was added a few drops of Meyers, Dragendorff’s and Wagner’s reagents respectively and
observed for appearance of yellowish or reddish-brown precipitate.
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3.5.2 Test for steroids:
About 2 mL of acetic anhydride was added to 1 mL of the extracted cellulose nanocrystals
colloidal solution. The mixture was boiled, cooled and 2 mL of sulphuric acid was added. A
brown ring at the joint of the two layers and the upper layer changing green indicates the
existence of steroids.
3.5.3 Test for Flavonoids (NaOH Test)
About 2 mL of the colloidal solution of the extracted cellulose nanocrystals was put in a test tube
and added 5 mL of the diluted sodium hydroxide followed by addition of 5 mL of dilute
hydrochloric acid. A yellow solution with NaOH turns colorless with dilute HCl which indicates
the presence of flavonoids (Table 3.2).
3.5.4 Test for Polyphenolic compounds
About 1 mL of the colloidal solution of the extracted cellulose nanocrystals was put in a test tube
and freshly prepared 10 % FeCl3 solution was added and observed for dark green coloration.
3.5.5 Test for saponins
A little amount of the extract was added to 4 mL of water and the resulting solution was shaken
strongly and allowed to stand for about 10 minutes. Thick persistent foam indicates the presence
of saponins.
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3.5.6 Test for tannins
To about 2 mL of colloidal solution of the extracted cellulose nanocrystals in a test tube a freshly
prepared iron (III) chloride solution was added. Formation of a dark blue or greenish grey
coloration of the solution indicates the presence of Tannins.
3.5.7 Test for Terpenes
About 2 mL of extracted cellulose nanocrystals colloidal solution was put in a test tube and
heated to dryness and carbon tetrachloride (CCl4) was added followed by few drops of acetic
anhydride. Concentrated sulfuric acid was added in drops and observed for formation of red
solution.
3.5.8 Test for terpenoids
About 2 mL of colloidal solution of the extracted cellulose nanocrystals was put in a test tube
with 2 mL of chloroform. 1 mL of concentrated sulphuric acid was gently added along the wall
of the tube. The observation of the presence of the reddish brown color at the interface shows the
presence of terpenoids.
3.6 Characterization of nanocellulose crystals
FTIR spectroscopy, Optical microscopy, SEM, XRD and TGA were used to characterize the
extracted nanocellulose crystals.
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3.6.1 Optical microscopy analysis
The raw sample of palmae sp. and CNCs were mounted on a microscope slide and viewed with
Leica DMLM optical microscope. Bright images were detected and collected on a computer
screen. Process was repeated for acacia sp. samples.
3.6.2 SEM
The surface morphology and cross-section of the nanocellulose crystal standard and sample were
analysed on scanning electron microscope, SEM (Fei Quanta 250 FEG, USA). The samples were
sputter-coated with gold prior to examination. SEM images of pure CNCs were taken at an
accelerated voltage of 5.0 kV at three-dimensional plots for individual visualization effect.
Statistical experimental designs were constituted and analyzed using Design Expert 7.0 (Stat-
Ease, Inc., USA). All experiments were done randomly and independently according to DOE’s
(Design of Experiment) run order in triplicate and average value of responses were used. (Bilgi
et al., 2015)
3.6.3 XRD
The X-ray diffraction analyses were carried out with a Shimadzu diffractometer (XRD-6000,
USA) controlled at 40 kV and 30 mA with graphite filtered CuK (λ = 1.5433 Å) radiation. Data
were acquired on a 2θ scale from 5 to 40°. The crystalline index of cellulose, CIr, of the CNCs
was determined using Segal’s empirical method:
CIr(%) = [(I200 – Iam)/I200 ] x 100 (1)
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The I200 refers to the peak intensity corresponding to crystalline cellulose I, and Iam is the peak
intensity of the non-crystalline part. (Segal et al., 1959)
3.6.4 FT-IR
The FTIR of all samples were carried out using Shimadzu, IR Prestige-21, USA over an
absorbance range of 4000 – 500 cm-1. The Fourier transform infrared (FTIR) spectroscopy
determination was made with KBr pellets in the 500 - 4000 cm-1 region with a resolution of 4
cm-1 for 30 scans and 0.5 cm-1 interval. The mixture of KBr and sample were dried (100 °C, 1 h),
and the samples were prepared instantly before measurement. The background spectra were
collected, using spectroscopic grade KBr. (Barud et al., 2015; Lustri et al., 2015)
3.6.5 TGA
Thermogravimetric analyses of palmae sp. and acacia sp. nanocrystals were carried out in a
Pyris 1 thermal analyzer (Perkin Elmer, USA) at a temperature range of room temperature to 800
°C at a heating rate of 10 °C per minute. The thermogravimetric analysis was done to examine
the thermal stability composition, purity, decomposition temperature and absorbed moisture
content. It is usually run with a differential scanning calorimeter. In this analysis about 9 mg of
the sample was put in an aluminium oxide crucible and subjected to heating in a nitrogen
atmosphere. The rate of decomposition was detected as 5 oC/min. All samples were preserved
under an inert atmospheric nitrogen with a flow rate of approximately 20 mL/min. The derivative
of each TGA curve was derived using the program Origin 8.1 (OriginLab, USA). (Kiziltas et al.,
2015)
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3.7 Quality assurance (QA) and quality control (QC)
Experiments were reproduced two or more times to verify the precision of the modified
extraction procedure. A certified reference cellulose nanocrystals was analyzed simultaneously
with the samples to examine the accuracy of the instrumental analysis.
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CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
4.1 Alkali Treatment
Alkali treatment was carried out using 4 % wt of sodium hydroxide. In this process, the main aim
was to remove the hemicellulose component of the plant material. Since hemicellulose materials
are irregularly arranged, the hydroxyl ion penetrates the structure thereby breaking down the
acetyl and pulcoumaric chains or bonds of hemicellulose. The presence of the hemicellulose in
the raw palmae sp. and acacia sp. was established by a peak at 1730 cm-1 which is attributed to
vibrations of acetyl and uronic ester groups. After the base hydrolysis, the band at 1730 cm-1 in
the FTIR spectrum disappeared showing the removal of hemicellulose. Similarly, part of lignin is
also removed after the base treatment. This is evident in the disappearance of the 1244 cm-1 peak
in the FTIR spectrum which is attributed to a stretching vibration of C=O bond. In addition,
color of the palmae sp. and acacia sp. changed slightly; the colour of palmae sp. treated with 4
% wt NaOH changed from dark brown to yellow after complete washing to a pH range of 6.00 –
7.00 and the acacia sp. base treated hydrolysis changed from brown coloration to pale yellow.
This color change confirms a partial removal of the lignin since the aromatic groups in the lignin
are the color pigment in plant. It was observed that the base treatment swelled, cracked and
solubilized the fiber structure which enlarges the surface area of the polymeric components
making it easily hydrolysed and washed away resulting in defibrillation. After the base
hydrolysis the pH of the resultant solution is highly basic of approximately 13. The solution is
washed to a neutral pH of 6.00 – 7.00. The pH changes are a major factor in isolating cellulose
nanocrystals. 6.00 – 7.00 pH level indicates a complete removal of the hemicellulose and other
extractives (Ng et al., 2015). It is only at this pH that the next process can be carried out.
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Figure 4.1 below is the gradual change in color of the supernatant after the first five washing
using a centrifuge at 3500 rcf for 3 mins for each spin. It was observed that the first supernatant
was the darkest of the five. This could be due to the high concentration of the hemicellulose,
lignin and extractives in solution. From the fourth washing the color of the supernatant is
constant with varying pH-levels. Thus subsequent washing reduced the pH of solution to neutral
to get rid of any residual components of hemicellulose, lignin and extractives.
Figure 4. 1: Reaction mechanism of the base hydrolysis using sodium hydroxide
4.2 Bleaching Treatment
The color of the plant material changes gradually throughout the bleaching process. For the
palmae sp. the color change was from pink to dark-brown, then to yellow, pale yellow and white.
The most effective color change occurred during the bleaching process where all lignin which is
responsible for the color are removed (Kargarzadeh et al., 2012; Singh et al., 2017). Bleaching
for the plant species used was repeated once for effective and complete removal of lignin. The
acacia sp. which was yellow got converted to pale yellow and finally white. After the first
bleaching, the color change was pale yellow and the second bleaching completely removed the
lignin leading to a pure white cellulose. It was observed that the color pigment in the palmae sp.
is more than the acacia. This is because the color transition of both palmae sp. and acacia sp.
was different even though the treatments were the same. After the first bleaching, the color of the
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palmae sp. was yellow whereas the acacia sp. was pale yellow. Consequently, acacia sp. took
less time for the second bleaching to convert to pure white. The bleaching treatment is significant
because the presence of lignin interfere with the acid treatment process since the lignin forms a
thin layer cover over the cellulose which prevents breakdown of the cellulose into the smaller
nano-size particles.
4.3 Acid Treatment
The most effective part of the extraction process is the acid hydrolysis which breaks down the
non-crystalline part of pure cellulose leaving nanosized crystalline particles. The acid hydrolysis
process is most effective with a highly concentrated sulfuric acid. In this process a highly
concentrated sulfuric acid, 64 %wt was used. The hydroxonium ion, H+ hydrolyses the
glycosidic bond between the amorphous and crystalline medium to release the pure cyrstallilne
leaving the amorphous domain to be washed away via repeated washing with deionized water.
The acid hydrolysis process also further breaks down the cellulose particles into nanocrystals by
reducing their length and diameter. It is easier breaking through the amorphous region due to the
irregular arrangement of the fibers. However, the crystalline domain is closely and regularly
packed which prevents the H+ ions from hydrolyzing. Eventually, the hydroxyl, OHs on the
cellulose are substituted for by the sulfonated ion, O SH-3 . This interaction makes the surface of
the nanocrystals more reactive. This also enhances the uniform dispersion of CNCs in solution
due to inter and intra electrostatic force of repulsion in solution. In the reaction, the OH on the
carbon 6 is more susceptible to react with the sulfuric acid due to less steric hindrance. Thus, the
OHs on carbon 2 and carbon 3 are not affected by the sulfuric acid hydrolysis. Below is a brief
scheme of the acid hydrolysis;
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Figure 4. 2: Reaction mechanism of acid treatment using sulfuric acid
The crystallinity of the cellulose increases through the process. This is because, amorphous and
non-crystalline part of the plant materials is removed. Accordingly, Battista et al. reported that
the defect of the non-crystalline region is responsible for the transverse cleaving of the pure
cellulose which enhances the acid hydrolysis.
4.4 Phytochemical screening
Phytochemical screening of the raw palmae sp. and acacia sp. tested positive whereas the
extracted and standard CNCs tested negative indicating an effective base and acid treatments
coupled with bleaching.
Table 4.1 represents the results from the phytochemical screening tests for alkanoid,
polyphenols, flavonoid and sapponins in the palmae sp. samples and CNC standard sample
obtained from cotton. The test confirms the presence of lignin, hemicellulose, residual pectin,
nitrogenous compounds and ash in the raw samples. CNCs from acacia sp., palmae sp. and
standard from cotton tested negatives, suggesting that all the lignin, hemicellulose, the residual
pectin, nitrogenous bases and ash were thoroughly removed by base and bleaching treatment.
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Table 4. 1: Phytochemical screening of the raw palmae sp. and acacia sp., CNCs from
palmae sp. and acacia sp. and Standard CNC.
Sample Flavonoid Polyphenols Saponins Alkaloids
A-Raw + + + +
P-Raw + + + +
CNC-A - - - -
CNC-P - - - -
CNC-Standard - - - -
(+) phytochemical present; (–) phytochemical absent
4.5 Characterization of isolated CNCs
To further corroborate the pure crystals, the following characterization were carried out.
4.5.1 FTIR Analysis
The FTIR technique was used to examine the functional groups in the samples at various stages
of the extraction. Figure 4.3 and 4.4 show the FTIR spectra of the untreated, bleached,
hydrolysed and cellulose nanoparticles. Dominant spectra band at 3339 cm-1 corresponds to the
stretching vibration of -OHs of intra and intermolecular hydrogen bond whereas 710 cm-1 is
assigned to -OH out of plane bending. Changes in strength of the H-bonding during the
extraction process affected the intensity and width of the spectra (Meyabadi et al., 2014). The
change in intensity of the spectra band of the OH group is also as a result of changes in the
number of H-bonding during the hydrolysis process. The presence of the spectra band in the
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region 1639 – 1648 cm-1 in all the fiber shows the presence of the -OH of the absorbed water.
The absorbed moisture at the peak of 1640 cm-1 show spectra intensity differences in the
untreated, bleached, hydrolysed samples and nanoparticles. Larger surface area of the treated
samples contributes to the intense broad peak of the OH band. Also after the base hydrolysis
more OHs are produced in solution which contributes to intense broad peak of the OH band.
Throughout the process the OH band progressively increases in height/intensity and width as a
result of the excess OHs.
Peaks in the regions of 2900 and 2930 cm-1 refer to the antisymmetric and symmetric vibrations
of – CH2 aliphatic bonds or groups. Also peaks in the region of 1410 – 1420 cm
-1 are due to the –
CH2 scissoring motion in the cellulose. Similarly C-H bending vibration in the molecule is
shown in the region of 1338 – 1368 cm-1. 1317 cm-1 shows – CH2 wagging. Also the peak at the
1040 cm-1 represents the C-O-C ether group of pyranose ring stretching vibration in the
cellulose. The peak at the 890 – 896 cm-1 corresponds to the beta-glycosidic linkage in cellulose
(Soni & Mahmoud, 2015). In addition, a spectra band at 1150 – 1159 cm-1 represents C-C
stretching vibration. Intense peak at 1750 cm-1 designates C=O stretch of aldehyde which is
corresponds to the presence of hemicellulose and lignin. The peak at 1230 cm-1 also represents
aromatic rings of lignin. Similarly, the presence of functional groups such as methoxyl-O-CH3, C
-O-C and aromatic C=C at peaks in the region between 1830 and 1730 cm−1 were observed
(Reddy & Yang, 2005a, 2005b). 2905cm-1 peak represents the HCH and OCH out of plane
bending vibrations. It is classified as a crystalline absorption peak. Vibration at 1370 cm-1 is
designated as CH deformation of the cellulose (Meyabadi et al., 2014). Additionally the band at
1160 is attributed to the asymmetric bridge stretching vibration of the C1-O-C4.
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Figure 4. 3: FTIR of acacia sp. from various treatment processes
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Figure 4. 4: FTIR of palmae sp. from various treatment processes
The peak present at 1765–1715 cm−1 in the spectrum of the raw fiber is indicative of the
presence of C=O linkage and aromatic stretch of C=C which is characteristic of ferulic and p-
coumaric acids of lignin and ester acetyl and uronic acid groups of hemicellulose (E Abraham et
al., 2011; Eldho Abraham et al., 2013). Peak at 1508 cm-1 corresponds to an aromatic stretch of
C=C which is responsible for lignin. 1247 cm-1 also corresponds to C-O-C of aryl, alkyl and
ether group. C=O stretching of the acetyl and uronic ester groups of hemicellulose or the ester
linkage of carboxylic groups of ferulic and p-coumaric acids were also designated at 1735 cm-1
(Alemdar & Sain, 2008a, 2008b). Also there is non-crystalline absorption peak related to COC,
CCO and CCH twist modes as well as stretching vibrations at C5 and C6 for the peak at 889 cm
-1.
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1060 and 895 cm-1 corresponds to C-O stretching and C-H rocking respectively of the
carbohydrates. Bands at 1053 cm-1 and 1030 cm-1correspond to the C-OH stretching vibration of
secondary and primary alcohols of cellulose, respectively (Johnsy George et al., 2011). The band
at 1623 cm-1 is due to the stretching vibration of C=O groups of cellulose rings.
Decreasing intensity of spectra band at 1730 cm-1, 1556 cm-1and 1240 cm-1 shows the removal of
hemicellulose, lignin and extractives associated with cellulose (Costa et al., 2015). Appendix A –
D. The absence of absorbed moisture was confirmed by the peak at 1638 cm-1. It was discovered
that peak at 1738 cm-1 disappeared after base hydrolysis during the extraction process confirming
the successful removal of hemicellulose from the plant samples (Soni & Mahmoud, 2015).
Lignin was not absolutely extricated after alkali hydrolysis but disappeared completely after
bleaching. It was also observed that the C=O stretching vibration of the ether bond in the
hemicellulose disappeared after the alkali hydrolysis. This could be due to the effectiveness of
the base hydrolysis in scattering of the ester linked substances of the hemicellulose. Accordingly,
in a research by Chieng et al., (2017), variation in the peaks of the FTIR spectra at 2916 cm-1 (C-
H stretching), 1732 cm-1 (C=O stretching) and 1234 cm-1 (C-O stretching) showed that the base
hydrolysis entirely extricated hemicelluloses and lignin from the fiber surface (Chieng et al.,
2017). This was also confirmed by Larissa et al. in their research using corn stover. In this
research it was indicated that changes in the peak at 1731, 1556 and 1244 cm-1 of the FTIR
spectrum showed a removal of hemicellulose, and partly lignin (Costa et al., 2015). The results
were further confirmed by a second method called the Ammonium persulfate (APS). This was a
24 hour treatment of alkali hydrolysis, bleaching and acid hydrolysis using a concentrated
solution of ammonium persulfate. It was evidenced that the peaks in the CNCs from both
methods are virtually the same. Table 4.2.
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Table 4. 2: FTIR vibrational frequency and peak assignment for palmae sp. and acacia sp.
Sample Vibrational frequencies (cm-1) Peak assignment
Untreated 3344.94 OH stretch vibration
2923.14 C-H stretch vibration
1737.20 C=O stretch of aldehyde
1595.31 C=C stretch of aromatic
1421.92 C-H bending of alkanes
1371.72 C-H bending of alkanes
1232.10 C-O bending
1035.27 C-O bending of alcohols
Alkaline treatment 3339.14 OH stretch vibration
2849.14 C-H stretch vibration
1637.92 OH bending
1421.89 C-H bending of alkanes
1370.14 C-H bending of alkanes
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1109.25 C-O bending of ethers
1057.15 C-O bending of ethers
1033.90 C-O bending of ethers
Bleach treatment 3341.21 OH stretch vibration
2903.04 C-H stretch vibration
1638.29 OH bending
1370.38 C-H bending of alkanes
1160.65 S-O stretch & C-O of esters
1057.59 C-O bending of ethers
Acid treatment 3339.25 OH stretch vibration
2903.04 C-H stretch vibration
1639.12 OH bending
1316.59 C-O bending of esters
1160.26 S-O stretch & C-O of esters
1105.54 C-H bending of alkanes
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1054.88 C-O bending of ethers
1032.46 C-O bending of ethers
Dialyzed 3339.03 OH stretch vibration
2904.56 C-H stretch vibration
1638.65 OH bending
1316.86 C-O bending of esters
1160.41 S-O stretch & C-O of esters
1107.35 C-O bending of ethers
1056.32 C-O bending of ethers
1033.43 C-O bending of ethers
Table 4. 3: Vibrational frequency and peak assignment of palmae sp. using Amonium
persulfate method of extraction.
Sample Vibrational frequencies (cm-1) Peak assignment
Palmae sp. (raw) 3344.94 OH stretch vibration
2923.14 C-H stretch vibration
1737.20 C=O stretch of aldehyde
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1595.31 C=C stretch of aromatic
1421.92 C-H bending of alkanes
1371.72 C-H bending of alkanes
1232.10 C-O bending
1035.27 C-O bending of alcohols
CNC 3339.08 OH stretch vibration
2902.35 C-H stretch vibration
1638.08 OH bending
1316.65 C-O bending esters
1160.51 S-O stretch & C-O of esters
1106.61 C-O stretch
1055.21 C-O stretch & C-H rocking
1032.89 C-O stretch & C-H rocking
4.5.2 SEM
The effect of the treatments (alkali, bleaching and acid) on the structure of the CNCs was
investigated by the use of scanning electron microscopy. Figures 4.5, 4.6, 4.7, 4.8 and 4.9
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summarize the micrographs of the CNCs obtained from both samples, CNC - A and CNC - P.
SEM images revealed a change in the structure compared to the optical images of the untreated
samples. This shows that the treatments may have removed the amorphous compounds of
hemicellulose, lignin, pectin, saponins, gums, wax, and polysaccharides.
4.5.2.1 Acacia sp.
The scanning electron microscopy was used to analyse the morphology of the extracted CNCs.
Figure 4.5 and 4.6 display the micrograph of the various images obtained at different
magnifications. It was observed that the images of the cellulose fibers are large aggregates
comprising of slender particles with dimensions of approximately less than 10 µm. Thus
nanoparticles have been produced but agglomerated. This agrees with works done by Brinchi et
al. when nanocellulose were extracted from lignocellulosic biomass. (Brinchi et al., 2013). The
formation of the CNCs also confirms the successful removal of the hemicellulose, lignin, wax,
pectin and other impurities on the surface of cellulose. In Fig. 4.6, the particles are well dispersed
in solution after filtration followed by dialysis. Filtration removed the larger particles which may
have a residual components of homocellulose and lignin leaving a more crystalline particles.
Furthermore, the uniform dispersivity of the particles suggested a more sulfonated group of CNC
particles which repel each other due to the electrostatic repulsive interaction of the negative
charge sulphonated ions in solution.
. .
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Figure 4. 5: SEM image of the CNCs from Acacia sp. (CNC - P, 1000x, scale bar = 100 µm)
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Figure 4. 6: SEM image of the cellulose nanocrystals from Acacia sp. after filtration
followed, followed by hydrolysis (CNC - P, scale bar = 10 µm)
4.5.2.2 Palmae sp.
The SEM images of the cellulose nanocrystals from the Palmae sp. are shown in Figure 4.7 and
4.8. Images of the cellulose nanocrystals in structure or morphology from the Palmae sp.
appeared to be similar to the Acacia sp. with few variations. However, the aspect ratio may differ
when the exact dimensions of the nanoparticles are determined with a transmission electron
microscope since the aspect ratio is the ratio of the length to width (Favier et al., 1995).
Appearance of the white images indicated the effectiveness of the treatment process during the
base hydrolysis and bleaching. This is because folded ribbon-like shape in the form of bundles
(Abe & Yano, 2009) as seen in the optical image of the raw sample was absent in the CNCs.
Like in the Acacia sp., the particles are approximately less than 10 µm with less agglomeration.
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This suggests a more effective acid hydrolysis in cleaving the 1,4-β-glycosidic linkage which
produced a more highly sulfonated CNCs particles which could disperse more uniformly in
solution due to the pronounced counter-ion interaction (Bondeson et al., 2006). In Figure 4.9
image, white protrusion called tyloses which is responsible for the presence of residual starch are
absent which is indicative of thethe successful removal of the starch making the CNCs more
pure. Additionally, the particles were seen not to be uniform in dimensions. This is also due to
the random cleaving of the glycoside bonds during the acid hydrolysis as well as the cracking of
the fasciculus which were held by the strong inter and intra molecular hydrogen bonding. The
degree of depolymerization may also have been reduced and eventually wide diverse range of
CNCs are produced due to the geographical locations and treatments processes (Bendahou et
al., 2009).
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Figure 4. 7: SEM image of the CNCs from palmae sp. (CNC - P, 400x, scale bar = 500 µm)
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Figure 4. 8: SEM image of the cellulose nanocrystals from palmae sp. after filtration,
followed by dialysis (CNC - P, scale bar = 10 µm)
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4.5.3 XRD
The X-ray diffraction analysis was carried out to examine crystallinity, thermal stability,
elasticity, absorptive capacity and other physical properties which may be vital to the industry.
Crystalline index which is the ratio of the crystalline to the non-crystalline region of cellulose
was used to examine the crystallinity of all samples. An increase in crystallinity index signifies
high strength due to high stiffness and rigidity. This is also indicative of high resistance to cracks
(Chieng et al., 2017). Peak intensity at 2θ value of 16o to 18o shows crystallinity of the
amorphous arrangement whereas 22o to 23o is related to the crystalline form of cellulose. Within
the limit of experimental errors peak intensity values at 22-23o are supposed to increase whilst
those at 16-18o decrease due to the removal of the non-crystalline part of cellulose. Figure 4.11
shows the X-ray diffractograms of acacia sp. (a) untreated (b) bleached (c) acid hydrolyzed and
(d) CNC. Table 4.4 presents a summary of the crystallinity index of the various stages.
Figure 4. 9: The X-ray diffractograms of acacia sp. (a) untreated (b) bleached (c) acid
hydrolyzed and (d) CNC.
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Table 4. 4: Crystallinity index (%) of untreated, base treated, bleached, acid hydrolyzed
and CNC of acacia sp.
2θ (Amorphous) (◦) 2θ(002)(◦)
Sample Degree Intensity (Iam) Degree Intensity (I002) CrI (%)
Untreated 18.15 121 22.55 46 -163.04
Base hydrolyzed 18.15 136 22.55 52 -161.54
Bleached 18.15 45 2255 101 55.45
Acid hydrolyzed 18.15 270 22.55 193 -39.90
CNC 18.15 86 22.55 28 -207.14
The crystallinity was calculated utilizing the equation
CIr(%) = [(I200 – Iam)/I200] x 100
Where l200 is the height of the peak at 2Ꝋ=22.5
o referring to the crystalline and amorphous
fraction and Iam is the height measured at 2Ꝋ=18
o referring to the amorphous fraction (Ahmadi et
al., 2015).
4.5.3.1 Untreated Acacia sp.
The untreated acacia sp. gave the second lowest crystallinity index of -163.04 %. However, this
was expected to be the lowest since it has the greatest amount of amorphous cellulose as well as
lignin and hemicellulose. From Figure 4.9, peaks at 18.15 and 22.55o were approximately absent
due to the numerous amorphous components and impurities associated with the cellulose.
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4.5.3.2 Base hydrolysis
Crystallinity index of the alkaline treated acacia sp. increased to -161.54 %. This was expected
but not in the negatives as reported by Chieng et al. in their work using Oil Palm Front. This also
shows partial removal of hemicellulose associated with the cellulose (Chieng et al., 2017).
4.5.3.3 Bleaching
From Figure 4.9, the peak intensity at 2θ value of 22.55o appeared after bleaching showing the
presence of crystalline component of the cellulose. However, the amorphous peak value at 18.15o
didn’t appear. This explains the increase in the crystallinity index after bleaching of the acacia
sp. Thus the bleaching was effective in removing few amorphous component associated with the
cellulose.
4.5.3.4 Acid hydrolysis
The acid hydrolyzed curve shown in Figure 4.9 indicates two main peaks at 18.15o and 22.55o.
However, the crystallinity index as summarized in Table 4.6 indicated a decrease in the
crystallinity. Decrease in the crystallinity index after acid treatment shows that the sulfuric acid
hydrolysis was not effective in breaking the amorphous component of cellulose away. Thus the
hydrolytic cleavage of the glycosidic bond was not too successful. This could be due to the
working environment in terms of temperature, concentration of acid and time of hydrolysis.
4.5.3.5 Cellulose nanocrystals
The crystallinity index computed for the isolated CNCs was -207.4%. This is the lowest of all the
crystallinity index values of the acacia sp. Additionally, the diffractogram of the CNC showed
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almost no peak at the 18o and 22o. These findings contradict what is reported in the FTIR results
and literature. For example in the isolation of CNCs from Menkuage leaves and Pineapple
biomass, the crystallinity index values increased gradually from the untreated plant material to
the isolated CNCs (Cherian et al., 2011; Sheltami et al., 2012).
4.5.3.6 Palmae sp.
Table 4. 5: Crystallinity index (%) of untreated, bleached, acid hydrolyzed and CNC from
Palmae sp.
2θ(Amorphous)(◦) 2θ(002)(◦)
Sample Degree Intensity (Iam) Degree Intensity (I002) CrI (%)
Untreated 18.2 84 22.9 68 -23.53
Bleached 18.2 52 22.9 137 62.04
Acid hydrolyzed 18.2 90 22.9 156 42.30
CNC 18.2 1390 22.9 25 -5460
4.5.3.7 Untreated
The untreated palmae sp. showed a very low crystallinity index of -23.53%. This is second to
that of the CNCs produced. Although from Figure 4.10 there isn’t any peak at the 18o and 22o
showing crystallinity. This is ascribed to the presence of numerous amorphous component
coupled with lignin and hemicellulose covering the crystalline cellulose in the plant material.
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4.5.3.8 Bleaching
XRD of the bleached palmae sp. exhibited a sharp peak at 22o indicative of the presence of
crystalline cellulose structure of 1,4-β-glycosidic linkages. From Table 4.5, the crystallinity of
the cellulose increased tremendously to 62o. The increase can be credited to the effective
removal of lignin, hemicellulose and other impurities leaving pure cellulose.
4.5.3.9 Acid hydrolysis
Crystallinity index after acid hydrolysis was 42.30%. This percentage suggest a higher
crystallinity but a decrease in value from bleaching treatment. The XRD results show that the
crystalline cellulose structure was not maintained during acid treatment. With Wang et al. who
reported that the crystal structure of cellulose was conserved after acid treatment, it was expected
that the crystallinity index increased progressively such that the CNCs gives the highest
crystallinity index (X. Wang et al., 2011).
4.5.3.10 Cellulose nanocrystals
The diffraction intensity peak at 2Ꝋ=18o which is related to the non-crystalline component of
cellulose was very strong for the CNCs produced (Ahmadi et al., 2015). However there was no
peak at 2Ꝋ=22o which correspond to the crystalline sections of cellulose. This culminated to the
CNC having the lowest crystallinity index of -5460% which could be treated as an outlier. The
results suggest that the treatment processes, base hydrolysis, bleaching and acid hydrolysis were
not too successful extracting and isolating the CNCs. Temperature variation, concentration of
base and acid as well time of reactions could also account for the not too good XRD results.
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However, from Figure 4.11, it was observed that the palmae sp. was more crystalline than the
acacia sp.
Figure 4.10: The X-ray diffractograms of palmae sp. (a) untreated (b) bleached (c) acid
hydrolyzed and (d) CNC.
1400
700
CNC-P
0 CNC-A
10 20 30 40 50
2Theta [Degree]
Figure 4. 11: X-ray diffractograms CNC from palmae sp. (P) and acacia sp. (A).
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Intensity [a.u]
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4.5.4 TGA
Usually, the TGA curve is divided into five major temperature components. First is temperature
below 150 oC which is due to loss of moisture, lower molecular weight solvents and desorption.
Next is the temperature between 150 and 250 oC. This is responsible for the loss of lower
molecular weight compounds such as additives, crystallization of water plasticizers. The third
temperature range is 250 oC and 500 oC which is responsible for decomposition of inert gasses or
oxidation of certain organic matter or simultaneous degradation processes such as
depolymerization, dehydration, and degradation of the glycosyl rings and later formation of a
charred remnants. Temperatures above 500 oC cause carbonization of hydrocarbonated
compounds which pyrolysis has no volatile formation. Beyond 500 oC is the decomposition of
metallic oxides or inorganic salts, ashes and also oxidation and breakdown of the burnt residue
into gaseous products with low molecular weight (Meyabadi et al., 2014)
TGA study was done at each stage of the extraction process and material was characterized by
measuring the change in mass as a function of temperature. Degradation patterns appeared to be
the same for all samples. It was observed that the decomposition peak temperature increased
gradually from the base hydrolysis to bleaching, and acid hydrolysis. This was expected because
in each stage there was a removal of various components of impurities like hemicellulose, lignin,
mucilage, wax, pectin, gums, tannins, proteins and starch. In the analysis, two decomposition
temperatures were observed. The first decomposition was loss of moisture whilst the second was
organic matter (Chartas et al., 2001). This could be due to the evaporation of surface moisture
and inside moisture by chemisorbed of the fiber sample which was confirmed by the moisture
detected by the FTIR peak at 1640 cm-1, a bending vibration of water intermolecular hydrogen
bonding. Also, Lignin decomposed at 200 oC and hemicellulose at 220 oC. Additionally,
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degradation at 231 oC is due to hemicellulose and 317 oC due to alpha cellulose 1. Here the
major characterization parameter of the material is the weight loss, residual weight,
decomposition peak, onset and maximum temperatures. The amount of mass loss was computed
as
m (%) = [ (mo- mt) / mo ] x 100 %
where m is the mass loss
mo is the initial mass and
mt is the final mass after decomposition
The parameters that characterize the samples are mass loss, mass residue, decomposition peak,
onset and end temperature.
4.5.4.1 Acacia sp.
The Figure 4.12 below shows the thermogram of the raw Acacia sp. before the treatment
processes. Moisture loss at the first decomposition was below 275.9 oC with a small weight loss
of 10%. It is expected that other components such as hemicellulose were included in the 10%
since hemicellulose decomposes at 170 oC (Chieng et al., 2017). The decomposition temperature
range was 275.9 oC to 367.6 oC. Of the 10.5 mg mass used in the analysis, the mass change was -
71.77% with a residual mass of 28.17% at 899.2 oC. During this temperature change organic
matter such as starch, pectin, protein and mucilages are decomposed. The residual temperature
which is the temperature at which no more organic matter is lost continued up to 899.2 oC. Here
decomposition of inorganic compounds at a temperature of 650 oC may occur. An example is a
conversion of wedelite to calcium oxide, CaO (Frost & Weier, 2003). Peak decomposition
temperature was 323.9 oC which was relatively low. The weight percentage of the char,
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carbonaceous residue beyond the 500 oC was 28.17%. This was the lowest instead of being the
highest which suggest the presence of few or impurities.
4.5.4.2 Base hydrolysis
The onset decomposition temperature after the alkali treatment (Ton) increased to 325
oC with a
maximum rate of decomposition, (Tmax) at 387.7
oC. This is evidenced in the removal of
hemicellulose and some aspect of wax and pectin during the base hydrolysis as seen in the FTIR
analysis. Percentage of moisture loss was supposed to be less but was still appreciable of about
10% even though hemicellulose was lost since it decomposes at a 170 oC and hence the mass.
Onset temperature rose from the 275.9 oC in the raw sample to 325.0 oC. This result shows that
the alkaline hydrolysis could eventually increase the thermal stability of the sample.
4.5.4.3 Bleaching
The onset temperature for the bleached sample decreased to 273.9 oC with a maximum
decomposition temperature of 387.7 oC. Apparently, the onset temperature was supposed to have
increased with the maximum decomposition temperature. This could be due to the presence of
some impurities and inorganic mineral salts. It could also mean that the bleaching was not too
effective in removing the xylan or lignin component of cellulose (Tang et al., 2015). This result
almost agrees with the XRD peak obtained after bleaching meaning that the crystallinity didn’t
really increase. The char weight percent increased from alkaline treated to the bleached which
suggests that the treatment process might have introduced some impurities into the cellulose.
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4.5.4.4 Acid hydrolysis
After the acid hydrolysis, the onset temperature decreased again to 273.9 oC with a
corresponding decrease in the maximum decomposition temperature at 355.6 oC. Thus instead of
having the thermal stability increased it rather decreased. This could be due to harsh treatment
process or condition not too effective. Additionally, the modification of the concentrations of the
acid used might have influenced the crystallinity. Howerver, this agrees with work done by Oun
et al. which showed that the use of sulfuric acid decreases the crystallinity due to the introduction
of the active sulphate group (Y. W. Chen et al., 2016). And one way to curb this occurrence as
reported by Oun et al. is to treat the acid hydrolyzed sample with 1M NaOH to diminish the
active effect of the sulphate. Similarly, the larger surface area of the nanoparticles which make
them more exposed to faster thermal change may also account for these changes. In addition, the
larger surface area causes faster heat transfer and hence lowering the thermal stability. It is
reported that the activation energy of the acid hydrolysed sample was minimized resulting in low
thermal stability.
4.5.4.5 Cellulose nanocrystals
Onset temperature and maximum decomposition temperature increased to 301.5 and 363.8 oC
respectively in the final CNCs. This could be due to the fact that the CNCs became more dense
and compact after successive removal of the amorphous component of the plant species (Usha et
al., 2016). In addition, the impurities associated with the cellulose which could have accelerated
the thermal decomposition may have been removed (Chirayil et al., 2014). In addition, after the
dissolution of the amorphous components of the cellulose the crystals structure rearranged and
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reorients to give a high composition of crystalline domain. This agrees with work done by
Mandal et al. (Mandal & Chakrabarty, 2011).
The residual weight beyond 500 oC could be carbonaceous residues resulting from carbonization
of cellulose under the nitrogen atmosphere. This carbonaceous residues, popularly known as char
could result from pyrolysis. This is because more ordered pairs of cellulose fibers require more
energy to degrade, resulting in the partial change in the weight percent. The weight percentage of
the char was given as 28.62 %.
Table 4. 6: Amount of Weight loss (%) and Charred residue (%) for different samples of
Acacia sp.
Sample Weight Loss Char yield (%)
Untreated -71.77 28.17
Base hydrolyzed -67.27 32.66
Bleached -64.23 35.75
Acid hydrolyzed -68.13 31.85
Cellulose nanoparticles -71.36 28.62
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Figure 4. 12: Thermogram of acacia sp., alkaline treated, bleached, acid hydrolysed and
CNC
4.5.4.6 Palmae sp.
The onset temperature for the untreated palmae sp. was 277.4 oC with a maximum
decomposition temperature at 366.4 oC. This is shown in Figure 4.15. Below 250 oC is the
decomposition of hemicellulose at about 170 oC or 220 oC and the loss absorbed moisture
(Chieng et al., 2017). This first decomposition gives a weight loss of approximately 10%. Out of
the 11.0 mg sample used, the weight loss at the decomposition temperature was 72.61% leaving
a residual mass of 27.24% at the residual temperature of 899.2 oC. Compared to other treatment
stages the untreated palmae sp. gave the highest mass change. And this agrees with work done
by Moran et al. (Morán et al., 2008). Thus the untreated was evidenced to have contained a lot of
impurities as well as non-cellulosic materials associated with the pure cellulose.
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4.5.4.7 Bleaching
The onset temperature for the bleached sample increased to 283.9 oC with a maximum
decomposition temperature of 308.2 oC. Apparently, the onset temperature was supposed to
increase with the maximum decomposition temperature. However, the maximum decomposition
temperature dropped quite significantly. This could be due to the presence of some impurities
and inorganic mineral salts. It could also mean that the bleaching was not too effective in
removing the xylan or lignin component of cellulose. The char weight percent which was
recorded as residual mass (%) increased greatly from the untreated 27.24 % to 41.21% after
bleaching. This is indicative of the fact that more carbon compounds were present together with
other inorganic material like CaO.
4.5.4.8 Acid hydrolysis
For the acid hydrolysis, the onset temperature increased to 287.4 oC with a corresponding
increase in the maximum decomposition temperature at 308.8 oC. This result indicates that the
thermal stability of the cellulose nanocrystals increased through the process treatments. Thus the
treatments are seen to be effective. However, this is contrary to some works which reported that
the use of sulfuric acid decreases the crystallinity due to the introduction of the active sulphate
group (Y. W. Chen et al., 2016). It also means that the particles became more orderly arranged
and became more compact and hence highly resistant to temperature changes.
4.5.4.9 Cellulose nanocrystals
The onset temperature of the Palmae sp. increased from 308.2 oC to 363.7 oC. Residual mass
decreased to 27.92 % at residual temperature of 899.3 oC. The decomposition peak temperature
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was also very high to a level of 336.3 oC. These increase in temperature of the CNCs show high
thermal stability and crystallinity. Meaning the cellulose nanocrystals produced has unique
physical properties of high resistance to heat. Additionally, the chemical properties of individual
atoms in the CNCs are seen to reorient and rearrange orderly making them look dense and more
compact. In a related study using the sulfuric acid by Chirayil et al. the thermal stability
increased (Chirayil et al., 2014). It also suggests that the impurities like pectin, wax and protein
were effectively removed. The non-cellulose amorphous part also was seen to have been
removed effectively leaving a more crystalline particle which is resistant to heat (Mandal &
Chakrabarty, 2011)
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Figure 4. 13: Thermogram of palmae sp., bleached, acid hydrolysed and CNC
Table 4. 7: Amount of Weight loss (%) and Charred residue (%) for different samples of
Palmae sp
Sample Weight Loss (%) Char yield (%)
Untreated -72.61 27.24
Base hydrolyzed -68.27 30.67
Bleached -58.78 41.21
Acid hydrolyzed -63.03 28.96
Cellulose nanoparticles -72.08 27.92
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CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATION
5.1 CONCLUSION
The main aim of this work was to investigate the viability of the palmae sp. and acacia sp. as a
local source for the isolation of nanocellulose crystals. This aim was achieved with the
successful isolation of CNCs from both plant biomasses.
Although harsh alkaline and acid hydrolysis conditions are needed for the extraction of CNCs,
the processes are highly sensitive to temperature and duration. For instance, temperature beyond
80 oC for alkaline treatment and 45 oC for acid treatment tends to lead to the charring of CNCs
into dark particles. Thus, careful attention is needed in ensuring the successful isolation of
CNCs.
The width dimension of the CNCs was approximately 200 nm with few micrometers of length.
Thus nanosized particles were produced in one dimension signifying the effectiveness of base
hydrolysis and bleaching. However, acid treatment appeared not to have been effective resulting
in longer sheets of cellulose.
The findings from the study suggest that, acacia sp. and palmae sp. may serve as good sources
for the production of nanocellulose crystals for potential use in diverse applications.
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5.2 RECOMMENDATION
Quite clearly, the conditions of extraction affect the nature and quality of nanocrystals formed.
Thus, optimization of the isolation processes to produce high quality nanoparticles is highly
encouraged. In this regard, the identification of important local sources of cellulose is
encouraged.
Additionally, investigations into alternate approaches in the extraction and isolation process
aimed at utilizing more eco-friendly and cheaper reagents are highly recommended.
Finally, it is recommended that the CNCs will be used as reinforcement material in the
production of biodegradable plastics.
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APPENDICES
APPENDIX A
FTIR spectra of acacia sp.
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FTIR spectra of palmae sp.
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APPENDIX B
TGA THERMOGRAPHS
TGA of Acacia sp.
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103
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104
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105
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TGA of Palmae sp.
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107
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108
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109